Inducible Regulatory T-Cell Generation for Hematopoietic Transplants

ABSTRACT

The present invention provides methods and compositions for converting non-Tregs into Tregs. The converted Tregs are referred to as inducible Tregs (iTregs). The iTregs are useful for preventing, suppressing, blocking or inhibiting an immune response. For example the iTregs are useful for preventing rejection of a transplanted tissue in a human or other animal host, or protecting against graft vs host disease. The iTregs can also be used to treat autoimmune diseases.

BACKGROUND OF THE INVENTION

The mammalian immune system plays a central role in protectingindividuals from infectious agents and preventing tumor growth. However,the same immune system can produce undesirable effects such as therejection of cell, tissue and organ transplants from unrelated donors.The immune system does not distinguish beneficial intruders, such as atransplanted tissue, from those that are harmful, and thus the immunesystem rejects transplanted tissues or organs. Rejection of transplantedorgans is generally mediated by alloreactive T cells present in the hostwhich recognize donor alloantigens or xenoantigens.

The transplantation of cells, tissues, and organs between geneticallydisparate individuals invariably results in the risk of graft rejection.Nearly all cells express products of the major histocompatibilitycomplex, MHC class I molecules. Further, many cell types can be inducedto express MHC class II molecules when exposed to inflammatorycytokines. Additional immunogenic molecules include those derived fromminor histocompatibility antigens such as Y chromosome antigensrecognized by female recipients. Rejection of allografts is mediatedprimarily by T cells of both the CD4 and CD8 subclasses (Rosenberg etal., 1992, Annu. Rev. Immunol. 10:333). Alloreactive CD4+ T cellsproduce cytokines that exacerbate the cytolytic CD8 response toalloantigen. Within these subclasses, competing subpopulations of cellsdevelop after antigen stimulation that are characterized by thecytokines they produce. Th1 cells, which produce IL-2 and IFN-γ, areprimarily involved in allograft rejection (Mossmann et al., 1989, Annu.Rev. Immunol. 7:145). Th2 cells, which produce IL-4 and IL-10, candown-regulate Th1 responses through IL-10 (Fiorentino et., 1989, J. Exp.Med. 170:2081). Indeed, much effort has been expended to divertundesirable Th1 responses toward the Th2 pathway. Undesirablealloreactive T cell responses in patients (allograft rejection,graft-versus-host disease) are typically handled with immunosuppressivedrugs such as prednisone, azathioprine, and cyclosporine A.Unfortunately, these drugs generally need to be maintained for the lifeof the patient and they have a multitude of dangerous side effectsincluding generalized immunosuppression.

Peripheral blood contains a small population of T cell lymphocytes thatexpress the T regulatory phenotype (“Treg”), i.e., positive for both CD4and CD25 antigens. There are several subsets of Treg cells (Bluestone etal., 2003 Nature Rev. Immunol. 3: 253). One subset of regulatory cellsdevelops in the thymus. Thymic derived Treg cells function by acytokine-independent mechanism, which involves cell to cell contact(Shevach, 2002 Nature Rev. Immunol 2: 389). They are essential for theinduction and maintenance of self-tolerance and for the prevention ofautoimmunity (Shevach, 2000 Annu. Rev. Immunol. 18: 423-449). Theseregulatory cells prevent the activation and proliferation ofautoreactive T cells that have escaped thymic deletion or recognizeextrathymic antigens, thus they are critical for homeostasis and immuneregulation, as well as for protecting the host against the developmentof autoimmunity. Thus, immune regulatory CD4⁺CD25⁺ T cells are oftenreferred to as “professional suppressor cells.”

Naturally arising CD4⁺CD25⁺ Treg cells are a distinct cell population ofcells that are positively selected on high affinity ligands in thethymus and that have been shown to play an important role in theestablishment and maintenance of immunological tolerance to selfantigens. Deficiencies in the development and/or function of these cellshave been associated with severe autoimmunity in humans and variousanimal models of congenital or induced autoimmunity.

Treg cells manifest their tolerogenic effects directly via cell-to-cellcontact or indirectly via soluble factors. Although the suppressivemechanisms of these cells remain to be fully elucidated, blockade ofIL-2 expression in effector T cells (Teff), physical elimination of Teffcells, induction of tolerogenic dendritic cells (DCs) via CTLA-4/B7axis, and inhibition of Teff cells via TGF-β and IL-10 are some of themechanisms that have been implicated to date. It also has been shownthat reverse signaling through CTLA-4/CD80 into Teff cells plays animportant role in their inhibition by Treg cells. Similarly,interactions between CTLA-4 on Treg cells and CD80 on DCs can result inreverse signaling and upregulation of the indoleamine dioxygenase enzymethat is involved in tolerance via the regulation of tryptophanmetabolism.

Treg cells can also be generated by the activation of mature, peripheralCD4⁺ T cells. Studies have indicated that peripherally derived Tregcells mediate their inhibitory activities by producing immunosuppressivecytokines, such as transforming growth factor-beta (TGF-β) and IL-10(Kingsley et al., 2002 J. Immunol. 168: 1080; Nakamura et al., 2001 J.Exp. Med. 194: 629-644). Treg are have been described in the literatureas being hypoproliferative in vitro (Sakaguchi, 2004 Ann. Rev. Immunol,22: 531). Trenado et al. provided the first evaluation of thetherapeutic efficacy of ex vivo activated and expanded CD4⁺CD25⁺regulatory cells in an in vivo mouse model of disease (Trenado et al.,2002 J. Clin. Invest. 112(11): 1688-1696).

However, the inadequacy of isolation and expansion methods used for thegeneration of Treg cell lines has significantly interfered with advancesin the research on human Treg cells. Thus, there has been a need formethods of producing sufficient number of these Treg cells to permitcharacterization and to provide for safe and effective therapeutic usein human patients. There has also remained a need for large-scaleexpansion of human CD4⁺CD25⁺ T cells for clinical trials including, butnot limited to immunotherapy or immunosuppression of cancers,particularly solid tumor cancers. Equally important has been a need tosuppress in vivo alloresponses and autoimmune responses, such as,although not limited to, graft-vs-host disease (GVHD).

BRIEF SUMMARY OF THE INVENTION

The invention provides a method of generating an inducible T regulatorycell (iTreg). The method comprises contacting a non-Treg with an agentcapable of converting the non-Treg into an iTreg, wherein the iTreg isimmunosuppressive.

In one embodiment, the agent is selected from the group consisting of abreakdown product of tryptophan, an analog of a metabolic breakdownproduct of tryptophan, a tryptophan catabolite, a demethylating agent, ahistone deacetylase inhibitor (HDACi), an mTOR inhibitor, and anycombination thereof.

In another embodiment, the non-Treg is further contacted with TGFβ.

In another embodiment, the non-Treg is selected from the groupconsisting of CD4+, CD4⁺CD25⁻, CD4⁺CD25⁻45RA+ cell, and any combinationthereof.

In yet another embodiment, the iTreg is CD4⁺CD25⁺.

In one embodiment, the non-Treg is isolated from a sample obtained fromleukopheresis products, bone marrow, lymph tissue, thymus tissue, spleentissue, or umbilical cord tissue.

In another embodiment, the tryptophan catabolite is kynurenines.

In another embodiment, the mTOR inhibitor is rapamycin.

In one embodiment, the demethylating agent is selected from the groupconsisting of 5-aza-2′-deoxycitidine (decitibine), 5-Azacytidine, andany combination thereof.

In one embodiment, the HDACi is selected from the group consisting oftrichostatin A (TSA), suberoylanilide hydroxamic acid (SAHA), and anycombination thereof.

In another embodiment, the method of generating an inducible Tregulatory cell (iTreg) further comprises contacting the non-Treg with abead or artificial antigen-presenting cell (aAPC) expansion system priorto or simultaneously with said agent. In one embodiment, the beadcomprises anti-CD3 antibody and anti-CD28 antibody.

In another embodiment, the method of generating an inducible Tregulatory cell (iTreg) further comprises contacting the iTreg with abead or artificial antigen-presenting cell (aAPC) expansion systemsubsequent to contacting the non-Treg with the agent. In one embodiment,the aAPC comprises an immune stimulatory ligand and at least oneco-stimulatory ligand on its surface.

In one embodiment, the stimulatory ligand is a polypeptide selected fromthe group consisting of a major histocompatibility complex Class I (MHCclass I) molecule loaded with an antigen, an anti-CD3 antibody, ananti-CD28 antibody, an anti-CD2 antibody, and any combination thereof.

In another embodiment, the co-stimulatory ligand is selected from thegroup consisting of CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL,OX40L, ICOS-L, ICAM, CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM,lymphotoxin beta receptor, ILT3, ILT4, 3/TR6, a ligand that specificallybinds with B7-H3, and any combination thereof.

The invention provides a method for inhibiting alloreactive T cells. Themethod comprises contacting alloreactive T cells with an effectiveamount of iTregs.

The invention provides a method for inhibiting cytotoxic T-lymphocyte(CTL) activity. The method comprises contacting a cytotoxic T-lymphocytewith an effective amount of iTregs.

The invention provides a method for generating an immunosuppressiveeffect in a mammal having an alloresponse or autoimmune response. Themethod comprising administering to the mammal an effective amount ofiTregs.

In one embodiment, the mammal having an alloresponse or autoimmuneresponse follows tissue transplantation, and wherein the method forgenerating an immunosuppressive effect in a mammal further comprisessuppressing, blocking or inhibiting graft-vs-host disease in the mammal.Preferably, the mammal is a human.

The invention provides a method for preventing an alloresponse or anautoimmune response in a mammal. The method comprising administering tothe mammal, prior to onset of an alloresponse or autoimmune response, aneffective amount of iTreg to prevent said response.

In one embodiment, the mammal is treated prior to, at the time of, orimmediately after tissue transplantation, and wherein the method furthercomprises preventing onset of graft-vs-host disease in the mammal.

In one embodiment, the mammal is treated prior to, at the time of, orimmediately after tissue transplantation, and wherein the method furthercomprises blocking rejection of the transplanted tissue in the mammal.

The invention provides a method of treating a transplant recipient toreduce in the recipient an immune response against the transplant. Themethod comprising administering to a transplant recipient, an effectiveamount of iTregs to reduce an immune response against the antigen.

In one embodiment, the method of treating a transplant recipient toreduce in the recipient an immune response against the transplantfurther comprises administering to the recipient an immunosuppressiveagent.

In one embodiment, the iTregs are administered to the recipient prior tothe transplant, concurrently with the transplant, or subsequent to thetransplantation of the transplant.

BRIEF DESCRIPTION OF THE DRAWINGS

For the purpose of illustrating the invention, there are depicted in thedrawings certain embodiments of the invention. However, the invention isnot limited to the precise arrangements and instrumentalities of theembodiments depicted in the drawings.

FIG. 1, comprising FIGS. 1A and 1B, is a series of charts demonstratingthe effects of rapamycin on expansion of CD4⁺CD25⁺ Tregs. Expansion withanti-CD3/28 microbeads in the presence of IL-2 failed to generateuniformly suppressive cells (FIG. 1B). Although anti-CD3 mAb loaded K562cells modified to express an FcR (CD64) and CD86 (KT64/86) was superiorto anti-CD3/28 beads for expanding cells, a high level of suppressionwas not uniformly observed with either approach (FIG. 1A). Adding rapa(labeled as +) reduced mean expansion rates by 30-fold with beadsresulting in ≦10-fold mean expansion rates by day 14. Rapa added toKT64/86 driven cultures (FIG. 1A) reduced mean expansion by 10-fold andimproved suppression.

FIG. 2 is a chart demonstrating that culturing CD4⁺25⁻ T cells withallogeneic TLR9 activated allogeneic plasmacytoid dendritic cells (pDCs)leads to the generation of iTregs, CD4⁺25⁺FoxP3⁺ suppressor cells.

FIG. 3, comprising FIGS. 3A-3C, is a series of charts demonstrating thatiTregs are immunosuppressive. FIG. 3A demonstrates that iTregs (closed),but not T cells primed to TLR activated B cells (open squares), werepotently suppressive of a naïve MLR culture. It was observed that iTreggeneration was dependent upon the enzyme indoleamine 2,3-dioxygenase(IDO) since IDO inhibition by 1-methyl-trypt (1MT) prevented iTregconversion (FIG. 3B). FIG. 3C demonstrates that addition of KYN resultedin potent MLR suppression that was not blocked by the addition of 1MT.

FIG. 4 is a series of charts summarizing the results of culturingCD4⁺CD25⁻ cells in diluent, decitibine, or tyrp/KYN. Diluent culturedcells expanded about 30-fold, decibine about 11 fold and low tryp/KYNabout 5-fold; CD4+CD127loFoxP3hi cells were 1%, 16%, and 13%,respectively. FoxP3 levels relate to suppressor potency and stability.iTreg suppression of CD8+ T cell proliferation (1:16 ratio), assessed byquantifying CFSE dye-dilution, was 18%, 41% and 76%, respectively,indicating that FoxP31nt cells present in the low trypt/KYN group werecontributing to suppressor cell potency.

FIG. 5, comprising FIGS. 5A-5F, is a series of charts summarizing theresults of culturing CD4⁺CD25⁻ cells in diluent, a demethylating agent,an HDACi, or a combination of a demethylating agent and an HDACi. FIG.5A summarizes the results of culturing CD4⁺CD25⁻ cells in diluent, TSA,TSA/decitibine, or SAHA/decitibine. It was observed in cultures with day3 TSA or SAHA that addition of decitibine on day 7 (SAHA/decitibine orTSA/decitibine) markedly increased CD4+127loFoxP3hi cells by day 10 from4.6% (diluent) to 44.5% (SAHA/decitibine), indicating that histoneacetylation followed by demethylation was advantageous for iTreggeneration (FIG. 5A). No iTreg generation was observed when the cellswere cultured in HDACi alone (FIGS. 5B and 5C; SAHA and TSA,respectively), FIG. 5D summarizes the results using 5-Aza-deoxycytidine(decitibine) in a bead- and cell-based antigen-presenting cell system.FIG. 5E summarizes the results using 5-azacytidine in a bead- andcell-based antigen-presenting cell system. FIG. 5F summarizes theresults using the combination of an HDACi with a demethylating agent ina bead- and cell-based antigen-presenting cell system.

FIG. 6 is an image demonstrating that re-stimulation of iTreg on day 7increases the percentage of Foxp3++ cells. nTreg (CD4⁺25⁺⁺) andCD4⁺25⁻45RA⁺ cells were purified from peripheral blood (PB) usingmagnetic beads and stimulated with anti-CD3 loaded KT64/86.Re-stimulation of CD4+25-CD45RA⁺ cells does not significantly effectFoxp3 levels, but the percentage of Foxp3++ are significantly increasedin the presence of Decitibine.

FIG. 7, comprising FIGS. 7A and 7B, is a series of images demonstratingthat TGFβ synergizes with Decitibine to induce Foxp3 expression. FIG. 7Ais a flow plot of CD4+ gated cells showing biphasic expression of Foxp3.FIG. 7B is an image summarizing the expression of Foxp3 after treatmentwith Decitibine in the presence and absence of TGFβ.

FIG. 8 is an image demonstrating that Tregs induced with Decitibine andTGFβ was observed to do not secrete IFNγ.

FIG. 9, comprising FIGS. 9A and 9B, is a series of images demonstratingthat Decitibine and Decitibine/TGFβ induced Tregs decrease mortality ina xenogeneic model of GVHD.

DETAILED DESCRIPTION

The present invention encompasses compositions and methods forconverting non-regulatory T cells (non-Tregs) into Tregs or converting amixed population of Tregs and non-Tregs into a substantially purifiedpopulation of Tregs. For example, the invention provides a method ofconverting CD4⁺CD25⁻ T cells into functional regulatory T cells. Theconverted cells are referred to as inducible Tregs (iTregs). In oneaspect, the iTregs are immunosuppressive.

In addition, the present invention provides a method for enhancingtolerance in a mammalian host to prolong foreign graft survival in thehost and for ameliorating inflammatory-related diseases, such asautoimmune diseases, including, but not limited to, autoimmunearthritis, autoimmune diabetes, asthma, septic shock, lung fibrosis,glomerulonephritis, artherosclerosis, as well as AIDS, and the like. Insome instances, the iTregs are useful for suppressing an immuneresponse.

The present invention further comprises a method for inhibitingproliferation of a T cell. Such inhibition can occur in vitro or invivo, preferably in an animal, more preferably in a mammal, even morepreferably in a human. This is because, as demonstrated by the datadisclosed herein, iTregs converted from non-Tregs according to themethods of the present invention are potent suppressors of T cellproliferation.

DEFINITIONS

As used herein, each of the following terms has the meaning associatedwith it in this section.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The term “about” will be understood by persons of ordinary skill in theart and will vary to some extent on the context in which it is used.

As used herein, to “alleviate” a disease means reducing the severity ofone or more symptoms of the disease.

“Allogeneic” refers to a graft derived from a different animal of thesame species.

“Alloantigen” is an antigen that differs from an antigen expressed bythe recipient.

The term “antibody” as used herein, refers to an immunoglobulinmolecule, which is able to specifically bind to a specific epitope on anantigen. Antibodies can be intact immunoglobulins derived from naturalsources or from recombinant sources and can be immunoactive portions ofintact immunoglobulins. Antibodies are typically tetramers ofimmunoglobulin molecules. The antibodies in the present invention mayexist in a variety of forms including, for example, polyclonalantibodies, monoclonal antibodies, Fv, Fab and F(ab)₂, as well as singlechain antibodies and humanized antibodies (Harlow et al., 1988; Houstonet al., 1988; Bird et al., 1988).

The term “antigen” or “Ag” as used herein is defined as a molecule thatprovokes an immune response. This immune response may involve eitherantibody production, or the activation of specificimmunologically-competent cells, or both. The skilled artisan willunderstand that any macromolecule, including virtually all proteins orpeptides, can serve as an antigen. Furthermore, antigens can be derivedfrom recombinant or genomic DNA. A skilled artisan will understand thatany DNA, which comprises a nucleotide sequences or a partial nucleotidesequence encoding a protein that elicits an immune response thereforeencodes an “antigen” as that term is used herein. Furthermore, oneskilled in the art will understand that an antigen need not be encodedsolely by a full length nucleotide sequence of a gene. It is readilyapparent that the present invention includes, but is not limited to, theuse of partial nucleotide sequences of more than one gene and that thesenucleotide sequences are arranged in various combinations to elicit thedesired immune response. Moreover, a skilled artisan will understandthat an antigen need not be encoded by a “gene” at all. It is readilyapparent that an antigen can be generated synthesized or can be derivedfrom a biological sample. Such a biological sample can include, but isnot limited to a tissue sample, a tumor sample, a cell or a biologicalfluid.

“An antigen presenting cell” (APC) is a cell that is capable ofactivating T cells, and includes, but is not limited to,monocytes/macrophages, B cells and dendritic cells (DCs).

The term “dendritic cell” or “DC” refers to any member of a diversepopulation of morphologically similar cell types found in lymphoid ornon-lymphoid tissues. These cells are characterized by their distinctivemorphology, high levels of surface MHC-class II expression. DCs can beisolated from a number of tissue sources. DCs have a high capacity forsensitizing MHC-restricted T cells and are very effective at presentingantigens to T cells in situ. The antigens may be self-antigens that areexpressed during T cell development and tolerance, and foreign antigensthat are present during normal immune processes.

The term “autoimmune disease” as used herein is defined as a disorderthat results from an autoimmune response. An autoimmune disease is theresult of an inappropriate and excessive response to a self-antigen.Examples of autoimmune diseases include, but are not limited to,Addision's disease, alopecia areata, ankylosing spondylitis, autoimmunehepatitis, autoimmune parotitis, Crohn's disease, diabetes (Type I),dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis,Graves' disease, Guillain-Barr syndrome, Hashimoto's disease, hemolyticanemia, systemic lupus erythematosus, multiple sclerosis, myastheniagravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoidarthritis, sarcoidosis, scleroderma, Sjogren's syndrome,spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxedema,pernicious anemia, ulcerative colitis, among others.

As used herein, the term “autologous” is meant to refer to any materialderived from the same individual to which it is later to bere-introduced into the mammal.

The term “cancer” as used herein is defined as disease characterized bythe rapid and uncontrolled growth of aberrant cells. Cancer cells canspread locally or through the bloodstream and lymphatic system to otherparts of the body. Examples of various cancers include but are notlimited to, breast cancer, prostate cancer, ovarian cancer, cervicalcancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer,liver cancer, brain cancer, lymphoma, leukemia, lung cancer and thelike.

A “disease” is a state of health of an animal wherein the animal cannotmaintain homeostasis, and wherein if the disease is not ameliorated,then the animal's health continues to deteriorate. In contrast, a“disorder” in an animal is a state of health in which the animal is ableto maintain homeostasis, but in which the animal's state of health isless favorable than it would be in the absence of the disorder. Leftuntreated, a disorder does not necessarily cause a further decrease inthe animal's state of health.

The term “DNA” as used herein is defined as deoxyribonucleic acid.

“Donor antigen” refers to an antigen expressed by the donor tissue to betransplanted into the recipient.

“Recipient antigen” refers to a target for the immune response to thedonor antigen.

As used herein, an “effector cell” refers to a cell which mediates animmune response against an antigen. An example of an effector cellincludes, but is not limited to a T cell and a B cell.

“Mixed lymphocyte reaction,” “mixed lymphocyte culture,” “MLR,” and“MLC” are used interchangeably to refer to a mixture comprising aminimum of two different cell populations that are allotypicallydifferent. At least one of the allotypically different cells is alymphocyte. The cells are cultured together for a time and undersuitable conditions to result in the stimulation of the lymphocytes,which in this particular invention are Treg cells. A frequent objectiveof an MLC is to provide allogeneic stimulation, such as may initiateproliferation of the Treg cells; but unless indicated, proliferationduring the culture is not required. In the proper context, these termsmay alternatively refer to a mixture of cells derived from such aculture. When cells from an MLC are administered as a bolus to a human,it is referred to as a “cellular implant.”

As used herein “endogenous” refers to any material from or producedinside an organism, cell, tissue or system.

By the term “effective amount”, as used herein, is meant an amount thatwhen administered to a mammal, causes a detectable level of immunesuppression or tolerance compared to the immune response detected in theabsence of the composition of the invention. The immune response can bereadily assessed by a plethora of art-recognized methods. The skilledartisan would understand that the amount of the composition administeredherein varies and can be readily determined based on a number of factorssuch as the disease or condition being treated, the age and health andphysical condition of the mammal being treated, the severity of thedisease, the particular compound being administered, and the like.

As used herein, the term “exogenous” refers to any material introducedfrom or produced outside an organism, cell, tissue or system.

The term “epitope” as used herein is defined as a small chemicalmolecule on an antigen that can elicit an immune response, inducing Band/or T cell responses. An antigen can have one or more epitopes. Mostantigens have many epitopes; i.e., they are multivalent. In general, anepitope is roughly about 10 amino acids and/or sugars in size.Preferably, the epitope is about 4-18 amino acids, more preferably about5-16 amino acids, and even more most preferably 6-14 amino acids, morepreferably about 7-12, and most preferably about 8-10 amino acids. Oneskilled in the art understands that generally the overallthree-dimensional structure, rather than the specific linear sequence ofthe molecule, is the main criterion of antigenic specificity andtherefore distinguishes one epitope from another. Based on the presentdisclosure, a peptide of the present invention can be an epitope.

The term “expression” as used herein is defined as the transcriptionand/or translation of a particular nucleotide sequence driven by itspromoter.

The term “expression vector” as used herein refers to a vectorcontaining a nucleic acid sequence coding for at least part of a geneproduct capable of being transcribed. In some cases, RNA molecules arethen translated into a protein, polypeptide, or peptide. In other cases,these sequences are not translated, for example, in the production ofantisense molecules, siRNA, ribozymes, and the like. Expression vectorscan contain a variety of control sequences, which refer to nucleic acidsequences necessary for the transcription and possibly translation of anoperatively linked coding sequence in a particular host organism. Inaddition to control sequences that govern transcription and translation,vectors and expression vectors may contain nucleic acid sequences thatserve other functions as well.

The term “helper T cell” as used herein is defined as an effector T cellwhose primary function is to promote the activation and functions ofother B and T lymphocytes and or macrophages. Most helper T cells areCD4 T-cells.

The term “heterologous” as used herein is defined as DNA or RNAsequences or proteins that are derived from the different species.

As used herein, “homology” is used synonymously with “identity.”

The term “immunoglobulin” or “Ig”, as used herein is defined as a classof proteins, which function as antibodies. The five members included inthis class of proteins are IgA, IgG, IgM, IgD, and IgE. IgA is theprimary antibody that is present in body secretions, such as saliva,tears, breast milk, gastrointestinal secretions and mucus secretions ofthe respiratory and genitourinary tracts. IgG is the most commoncirculating antibody. IgM is the main immunoglobulin produced in theprimary immune response in most mammals. It is the most efficientimmunoglobulin in agglutination, complement fixation, and other antibodyresponses, and is important in defense against bacteria and viruses. IgDis the immunoglobulin that has no known antibody function, but may serveas an antigen receptor. IgE is the immunoglobulin that mediatesimmediate hypersensitivity by causing release of mediators from mastcells and basophils upon exposure to allergen.

The term “immunostimulatory” is used herein to refer to increasingoverall immune response.

The term “immunosuppressive” is used herein to refer to reducing overallimmune response.

“Initiating iTreg conversion” as used herein refers to any event whichresults in a detectable increase in the phenotype and/or genotypecharacteristic of regulatory T cells. For example, a phenotype and/orgenotype characteristic of regulatory T cell is CD25 expression thusresulting in the generation of CD4⁺CD25⁺ cells from CD4⁺CD25⁻ cells.Another phenotype and/or genotype characteristic of regulatory T cell isimmunosuppression.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence. Thephrase nucleotide sequence that encodes a protein or an RNA may alsoinclude introns to the extent that the nucleotide sequence encoding theprotein may in some version contain an intron(s).

The term “polynucleotide” as used herein is defined as a chain ofnucleotides. Furthermore, nucleic acids are polymers of nucleotides.Thus, nucleic acids and polynucleotides as used herein areinterchangeable. One skilled in the art has the general knowledge thatnucleic acids are polynucleotides, which can be hydrolyzed into themonomeric “nucleotides.” The monomeric nucleotides can be hydrolyzedinto nucleosides. As used herein polynucleotides include, but are notlimited to, all nucleic acid sequences which are obtained by any meansavailable in the art, including, without limitation, recombinant means,i.e., the cloning of nucleic acid sequences from a recombinant libraryor a cell genome, using ordinary cloning technology and PCR™, and thelike, and by synthetic means.

The term “polypeptide” as used herein is defined as a chain of aminoacid residues, usually having a defined sequence. As used herein theterm polypeptide is mutually inclusive of the terms “peptide” and“protein”.

The term “self-antigen” as used herein is defined as an antigen that isexpressed by a host cell or tissue. Self-antigens may be tumor antigens,but in certain embodiments, are expressed in both normal and tumorcells. A skilled artisan would readily understand that a self-antigenmay be overexpressed in a cell.

As used herein, “specifically binds” refers to the fact that a firstcomposition binds preferentially with a second composition and does notbind in a significant amount to other compounds present in the sample.

As used herein, a “substantially purified” cell is a cell that isessentially free of other cell types. A substantially purified cell alsorefers to a cell which has been separated from other cell types withwhich it is normally associated in its naturally occurring state. Insome instances, a population of substantially purified cells refers to ahomogenous population of cells. In other instances, this term referssimply to cells that have been separated from the cells with which theyare naturally associated in their natural state. In some embodiments,the cells are culture in vitro. In other embodiments, the cells are notcultured in vitro.

As the term is used herein, “substantially separated from” or“substantially separating” refers to the characteristic of a populationof first substances being removed from the proximity of a population ofsecond substances, wherein the population of first substances is notnecessarily devoid of the second substance, and the population of secondsubstances is not necessarily devoid of the first substance. However, apopulation of first substances that is “substantially separated from” apopulation of second substances has a measurably lower content of secondsubstances as compared to the non-separated mixture of first and secondsubstances.

A “population” is used herein to refer to a group of cells having asubstantially similar phenotypic characteristic

“Transplant” refers to a biocompatible lattice or a donor tissue, organor cell, to be transplanted. An example of a transplant may include butis not limited to skin cells or tissue, bone marrow, and solid organssuch as heart, pancreas, kidney, lung and liver. A transplant can alsorefer to any material that is to be administered to a host. For example,a transplant can refer to a nucleic acid or a protein.

The term “T-cell” as used herein is defined as a thymus-derived cellthat participates in a variety of cell-mediated immune reactions.

The term “B-cell” as used herein is defined as a cell derived from thebone marrow and/or spleen. B cells can develop into plasma cells whichproduce antibodies.

As used herein, a “therapeutically effective amount” is the amount of atherapeutic composition sufficient to provide a beneficial effect to amammal to which the composition is administered.

As used herein, “treating” refers to the reduction, alleviation orelimination, preferably to normal levels, of one or more of the symptomsof the disease or condition which is being treated, e.g. alleviation ofimmune dysfunction or avoidance of transplant rejection, relative to thesymptoms prior to treatment. As used herein “treating” or “treatment”includes both therapeutic and prophylactic treatments.

The term “vaccine” as used herein is defined as a material used toprovoke an immune response after administration of the material to amammal.

A “vector” is a composition of matter which comprises an isolatednucleic acid and which can be used to deliver the isolated nucleic acidto the interior of a cell. Numerous vectors are known in the artincluding, but not limited to, linear polynucleotides, polynucleotidesassociated with ionic or amphiphilic compounds, plasmids, and viruses.Thus, the term “vector” includes an autonomously replicating plasmid ora virus. The term should also be construed to include non-plasmid andnon-viral compounds which facilitate transfer of nucleic acid intocells, such as, for example, polylysine compounds, liposomes, and thelike. Examples of viral vectors include, but are not limited to,adenoviral vectors, adeno-associated virus vectors, retroviral vectors,and the like.

“Xenogeneic” refers to a graft derived from an animal of a differentspecies.

As used herein, the term “CD127” refers to the alpha subunit of the“interleukin-7 receptor,” present on a Treg cell surface. The IL-7receptor alpha chain is described in the literature. See, e.g., Goodwinet al., 1990 Cell 60:941-951. CD127⁺ refers to cells which stainintensely or brightly when treated with a labeled antibody directedtoward CD127. CD127^(Lo/−) refers to cells of a type which stainsslightly/dully or not at all when contacted with a labeled CD127antibody. Generally, the cells are distinguished according to theirCD127 expression levels based upon a readily discernible differences instaining intensity as is known to one of ordinary skill in the art.

As used herein, the term “CD4” refers to a cell-surface glycoproteintypically found on the mature helper T cells and immature thymocytes, aswell as on monocytes and macrophages. CD4⁺ refers to cells which stainbrightly when contacted with labeled anti-CD4 antibody, and CD4⁻ refersto cells of a type which stain the least brightly, dull or not at all,when contacted with a fluorescently labeled CD4 antibody. Generally, thecells are distinguished according to their CD4 expression levels basedupon a readily discernible differences in staining intensity as the CD4staining is clearly bimodal.

As used herein, the term “CD25” refers to the alpha subunit ofinterleukin-2 receptor, a single-chain glycoprotein with a molecularweight of 55 kD. CD25^(hi) refers to cells which stain brightly whencontacted with labeled anti-CD25 antibody, CD25⁺ refers to cells whichstain less brightly when contacted with labeled anti-CD25 antibody, andCD25^(lo/−) refers to cells which are of a type which stains the leastbrightly, dull or null when contacted with a labeled CD25 antibody.Generally, the cells are distinguished according to their CD25expression levels based upon differences in staining intensity as isknown to one of ordinary skill in the art. In some embodiments, the cutoff for designating a cell as a CD25 expression category hi, +, lo, or −cell can be set in terms of the fluorescent intensity distributionobserved for all the cells. Generally, cells in the top 2, 3, 4, or 5%of staining intensity are designated “hi”, with those falling in the tophalf of the population categorized as being “+”. Those cells fallingbelow 50%, of fluorescence intensity are designated as CD25^(lo) cellsand below 5% as CD25⁻ cells.

Description

The present invention relates to methods and compositions for convertingnon-regulatory T cells (non-Tregs) into regulatory T cells. For example,the methods include converting CD4+ or CD4⁺CD25⁻ into CD4⁺CD25⁺ T cells.The constitutive expression of CD25 is considered to be a characteristicfeature of Tregs. Thus, Tregs are often CD4⁺CD25⁺ T cells, andpreferably immunosuppressive. Converted CD4⁺CD25⁺ T cells are referredherein as inducible Tregs (iTregs). In some instances, induction ofTregs includes both the generation of Tregs from naïve T cells and thereactivation of quiescent Tregs.

In some instances, the conversion process includes a combinationalapproach including an initial conversion stage, an outgrowth stage thatfavors Treg over non-Tregs, and an imprinting stage thereby generatingiTregs. The induction of Tregs is associated with the induction ofimmune tolerance and the suppression of an immune response.

In one embodiment, iTreg conversion includes subjecting non-Tregs to anamino acid starvation environment. For example, initiation of iTregconversion can be accomplished by exposing non-Tregs with a tryptophandepletion/catabolite.

The present invention also provides a method of reproducing the effectsof indoleamine 2,3 dioxygenase (IDO) to induce iTreg conversion by usinga downstream tryptophan metabolite, including, but not limited tokynurenin (also referred to herein as “KYN” or “kyn”).

In one embodiment, iTreg conversion includes subjecting non-Tregs to ademethylating agent.

In one embodiment, iTreg conversion includes subjecting non-Tregs to aDNA methyltransferase inhibitor.

In some instances, the combinational approach also includes incubatingthe T cells with an agent that is capable of favoring Treg conversionand outgrowth. An example of such an agent is rapamycin. Rapamycin is animmunosuppressive agent used to prevent allograft rejection. Rapamycinhas been shown to selectively expand naturally occurring CD4⁺CD25⁺FoxP3⁺regulatory T cells. The present invention provides a new protocol usingRapamycin to generate new Treg cells from non-Tregs (e.g., CD4⁺CD25⁻ Tcells). These inducible Tregs generated in accordance with the methodsof the invention exhibit immunosuppressive capacities.

In other instances, the combinational approach also includes incubatingnon-Tregs with a demethylating agent and histone deacetylace inhibitor(HDACi) for inducing iTregs conversion. In other instances, the approachincludes incubating the T cells with a demethylating agent and/or ahistone deacetylace inhibitor (HDACi) for imprinting iTregs after theconversion as been initiated.

In some instances, the combinational approach also includes incubatingnon-Tregs with a demethylating agent (or a DNA methyltransferaseinhibitor) and TGFβ for inducing iTregs conversion.

In a preferred aspect, the present invention provides a combinationalapproach for converting non-Tregs (e.g., CD4⁺CD25⁻ T cells) comprisingany one or more of the following steps: (i) isolating non-Tregs from asample (ii) contacting the cells with a) an agent that promotes iTregconversion, (iii) incubating the cells under conditions to allowproliferation and (iv) isolating the T cells after the incubation.

The invention also provides methods and compositions for ex vivoconversion and expansion of Tregs from non-Tregs. The expansion methodsfor iTregs generally comprise the use of a bead- or cell-basedartificial antigen-presenting cell. However, any method in the art canbe used to expand the iTregs.

The present invention provides a method of large-scale conversion andexpansion of iTregs that addresses the low numbers of natural Treg cellsthat can be isolated and expanded. Thus, the methods and compositions ofthe invention are useful for therapeutic purposes, for example, in theprevention and treatment of immune-based disorders and in the preventionof allograft rejection.

Isolating and Inducible Treg Conversion

Naturally occurring regulatory T (Treg) cells suppress immune responsesand play an important role in immunotherapy against autoimmune diseasesand provide transplantation tolerance. Various populations of Treg cellshave been described and include naturally occurring CD4⁺CD25⁺FoxP3⁺cells. The natural occurring CD4⁺CD25⁺FoxP3⁺ Treg cells represents about5-10% of the CD4⁺ T cells in the peripheral blood and are in ahypoproliferative state which has hampered detailed characterization andthe potential use of these cells in a therapeutic setting. It has alsobeen reported that about 1-2% of CD4⁺ T cells are CD25br natural Tregsin peripheral blood. In vivo uses therefore have relied on expansionprotocols to generate sufficient numbers of Treg cells for in vivo use.The clinical use of Treg cells is limited by the lack of appropriateisolation and expansions protocols to generate sufficient numbers for invivo infusion.

The present invention provides a method of generating a population ofimmunosuppressive Treg cells from the abundant CD4⁺CD25⁻ T cellpopulation. This method allows for the generation of Treg cells insufficient numbers for in vivo infusions. The method can be used bothfor generating Treg cells for research purposes and for clinical use byinfusion in patients.

In some embodiments, the invention provides for methods of selecting orisolating the cells so identified. In some embodiments, T cells areobtained from blood (e.g., isolated from PBMC), lymphoid, thymus or anyspecific tissues/organ sample of interest. These tissues or organs wouldinclude the pancreas, eye, heart, liver, nerves, intestine, skin,muscle, and joints.

The cells bearing the desired markers can be isolated, for instance, bythe use of labeled antibodies or ligands with FACS or magneticparticles/bead technologies as known to one of ordinary skill in theart. Accordingly, in some embodiments, the invention provides a methodof generating an isolated population of immunosuppressive regulatoryT-cells which are substantially CD4⁺CD25⁺ by obtaining a biologicalsample comprising non regulatory T-cells including, but not limited to,CD4⁺, CD4⁺CD25⁻, CD4⁺CD25⁻45RA+ cells, and converting the non regulatoryT cells into regulatory T cells or otherwise referred to as inducible Tcells (iTregs). In some embodiments, the population of convertedinducible T cells is substantially CD4⁺CD25⁺ T cells.

Non-Tregs, such as CD4⁺, CD4⁺CD25⁻, and CD4⁺CD25⁻45RA+ cells can beisolated by negative selection (e.g., CD8 and CD25). To enhanceenrichment of non-Tregs, positive selection may be combined withnegative selection against cells comprising surface makers specific tonon-Treg cell types, CD11b, CD16, CD19, CD36 and CD56-bearing cells.Preferably, a positive marker for positive selection is 45RA. As anon-limiting example, CD4⁺CD25⁻45RA+ cells can initially be isolated bynegative selection (e.g., CD8 and CD25). To enhance enrichment ofCD4⁺CD25⁻45RA+ cells, positive selection may be combined with negativeselection against cells comprising surface maker 45RA.

Sources of T cells and methods of isolating particular T cellpopulations (e.g. CD4⁺ cells) which can be converted by stimulationaccording to the methods of the present invention are well known anddescribed in the literature. Thus for example T cells may convenientlybe isolated from the blood e.g. from a peripheral blood mononuclear cell(PBMC) population isolated from blood, or from other blood-derivedpreparations such as leukopheresis products or from bone marrow, lymph,thymus, spleen or umbilical cord. T cell populations may be derived fromany appropriate source, including human or animal sources.

The present invention also includes a combinational approach ofgenerating regulatory T cells (Tregs) in vitro. The method includesobtaining non-Tregs (e.g., CD4⁺CD25⁻ cells) or mixed populations ofTregs and non-Tregs from a subject and converting the non-Tregs intoinducible Tregs. Converting non-Tregs into iTregs includes at least oneor more of the following stages: a stage of initiating the iTregconversion process; a stage to favor Treg conversion and outgrowth; anda stage for imprinting iTregs after conversion has been initiated. Anyexpansion method can be used in conjunction with the combinationalapproach of the present invention. For example, a bead- or cell-basedartificial antigen-presenting cell system can be used before, during,and/or after any of the stages included in the combinational approach ofgenerating Tregs.

Initiation Stage:

It has been shown that cells expressing the tryptophan-catabolizingenzyme, indoleamine 2,3-dioxygenase (IDO), are capable of inhibiting Tcell proliferation in vitro and reducing T cell immune responses invivo. The immunosuppressive effect of IDO can be blocked by the in vivoadministration of an IDO inhibitor, such as 1-methyl-tryptophan (alsoreferred to herein as 1-MT or IMT). IDO degrades the essential aminoacid tryptophan. IDO is the first and rate-limiting step in thedegradation of tryptophan to the downstream metabolite kynurenine (KYN)and subsequent metabolites along the KYN pathway. The results presentedherein demonstrate that conversion of non-Tregs into Tregs is partlyattributable to the biological activity of IDO, where the conversionprocess is partly dependent on the ability of IDO to catabolizetryptophan. Therefore, the present invention is partly based onIDO-mediated production of metabolites in the KYN pathway as theexemplary mechanism of Treg generation by the combinational approach ofthe invention.

The present invention demonstrates that IDO expression contributes tothe generation of CD4⁺ Tregs and demonstrates that this effect can bepharmacologically reproduced by the addition of a metabolic breakdownproduct of tryptophan, or an analog of a metabolic breakdown product oftryptophan. Tryptophan is also referred to herein as “Tryp,” “tryp,”“Trp” or “trp.”

The present invention includes methods of initiating conversion ofnon-Tregs into Tregs or otherwised referred to as inducible Tregs. Themethod includes contacting non-Tregs with a metabolic breakdown productof tryptophan, or an analog of a metabolic breakdown product oftryptophan. This stage of the combinational approach of the inventionincludes for example incubating CD4⁺ cells (non Tregs) in tryptophandepletion conditions for a time sufficient to induce the convesion ofnon-Tregs to Tregs. In some instances, the conversion process includesinduction of a stress response. Preferably, a stress response is createdby contacting non Tregs with trypt for a period of time followed byculturing the cells in additional permissive growth conditions. In someinstances, permissive growth conditions can be created using tyrptand/or typt metabolites. Preferrably, the permissive condition is thecombination of low concentrations of trypt and KYN (trypt/KYN).

The metabolic breakdown product of tryptophan, or an analog of ametabolic breakdown product of tryptophan, may be contacted withnon-Tregs in an amount effective to induce IDO expression. The resultspresented herein demonstrate that induced IDO activity stimulatesconversion of non-Tregs (e.g., CD4⁺CD25⁻) into Tregs to acquireincreased T cell suppressor functions. It is also believed that IDOactivity stimulates rapid increase of Treg suppressor functions andactivates the GCN2 stress response selectively in Tregs. The combinedeffects of Trp depletion and Trp catabolites induces non Tregs toacquire a regulatory phenotype, and that this mechanism is believed tobe mediated by GCN2. The protein kinase GCN2 is also referred to as“General Control Nonderepressible 2,” “eIF2AK4,” and “eukaryotictranslation initiation factor 2 alpha kinase 4”.

The present invention includes the use of a metabolic breakdown productof tryptophan, or an analog of a metabolic breakdown product oftryptophan, for the generation of Tregs. As used herein, an “analog”refers to a chemical compound or molecule made from a parent compound ormolecule by one or more chemical reactions. As such, an analog can be acompound with a structure similar to or based on that of a metabolicbreakdown product of tryptophan, but differing from it in respect tocertain components or structural makeup, which may have a similar actionmetabolically. In preferred embodiments, the metabolic breakdown productof tryptophan is L-kynurenine, kynurenic acid, anthranilic acid,3-hydroxyanthranilic acid, quinolinic acid, or picolinic acid, and ananalog of a metabolic breakdown product of tryptophan is an analog ofL-kynurenine, kynurenic acid, anthranilic acid, 3-hydroxyanthranilicacid, quinolinic acid, or picolinic acid.

Another approach to initiate the iTreg conversion process is to exposenon-Tregs with a demethylating agent or an inhibitor of DNA methylation.This is because contrary to non-Tregs, natural Tregs are hypomethylatedand it has been observed that complete demethylation and histonemodifications occur in the FoxP3 locus of natural Tregs. Therefore,exposing non-Tregs to a demethylating agent would alter the chromosomalstructure of the non-Tregs in a manner that renders the non-Tregs moresuseptiable for iTreg conversion. It is believed that exposing non-Tregsto a demethylating agent, such as 5-aza-2′-deoxycitidine (decitibine) or5-Azacytidine induces significant expression of FOXP3 and therebyinitiates the conversion of non Tregs into Tregs. Without wishing to bebound by any particular theory, decitibine is also considered to be aDNA methyltransferase inhibitor.

Another approach to initiate the iTreg conversion process is to exposenon-Tregs with a combination of a demethylation agent (or a DNAmethyltransferase inhibitor) and a histone deacetylase inhibitor(HDACi).

Another approach to initiate the iTreg conversion process is to exposenon-Tregs with a combination of a demethylation agent (or a DNAmethyltransferase inhibitor) and TGFβ.

Conversion and Outgrowth Stage:

Rapamycin (Rapa) is an immunosuppressive agent used to prevent allograftrejection. The cellular target for Rapamycin in vitro has beendiscovered, and was shown to selectively expand naturally occurringregulatory T cells (e.g., CD4⁺CD25⁺FoxP3⁺). The present inventionrelates to a combiantional approach of converting non-Tregs into Tregs(referred to as inducible Tregs; iTregs) using Rapamycin in combinationwith other agents and methods discussed elsewhere herein to optimize theconversion and large-scale expansion of iTregs. This is because theresults presented herein demonstrate that Rapa can be used to promoteselective outgrowth of Tregs over non-Tregs.

Rapa, an mTOR (mammalian target of rapamycin) inhibitor, has been shownto suppress non-Tregs and favor iTreg conversion. mTOR is a member ofthe PIK-related family of large protein kinases and mediates thephosphorylation of at least two regulators of protein synthesis and cellgrowth: S6 Kinase 1 (S6K1) and an inhibitor of translation initiation,the eIF-4E binding protein 1 (4E-BP1). mTOR is an important signalingintermediate molecule downstream of the PI3K/AKT pathway that inhibitsapoptosis, and is important in nutritional status checkpoint, mTOR is alarge multidomain serine/threonine kinase, and is a member of the PI3Kfamily of protein kinases based on homology within its catalytic domain.It has been shown that natural Tregs are less dependent than non-Tregson the mTOR/Akt pathway. Therefore, exposing non-Tregs to Rapa favorsiTregs and natural Tregs due to the dependency of non-Tregs onmTOR/p-Akt for proliferation and survival. Accordingly, the inventionincludes the use of any mTOR inhibitor to selectively favor theoutgrowth of iTregs and/or natural Tregs over non-Tregs.

Mammalian target of rapamycin (“mTOR”) regulates the activity of atleast two proteins involved in the translation of specific cell cycleregulatory proteins. One of these proteins, p70s6 kinase, isphosphorylated by mTOR on serine 389 as well as threonine 412. Thisphosphorylation can be observed in growth factor treated cells byWestern blotting of whole cell extracts of these cells with antibodyspecific for the phosphoserine 389 residue. As used herein, the term“mTOR inhibitor” means a compound or ligand which inhibits cellreplication by blocking progression of the cell cycle from G1 to S byinhibiting the phosphorylation of serine 389 of p70s6 kinase by mTOR.One skilled in the art can readily determine if a compound, such as arapamycin derivative, is an mTOR inhibitor.

As used herein, the term “rapamycin derivatives” includes compoundshaving the rapamycin core structure as defined in U.S. patentapplication Publication No. 2003/0008923, which may be chemically orbiologically modified while still retaining mTOR inhibiting properties.Such derivatives include esters, ethers, oximes, hydrazones, andhydroxylamines of rapamycin, as well as compounds in which functionalgroups on the rapamycin core structure have been modified, for example,by reduction or oxidation. Pharmaceutically acceptable salts of suchcompounds are also considered to be rapamycin derivatives.

Specific examples of esters and ethers of rapamycin are esters andethers of the hydroxyl groups at the 42- and/or 31-positions of therapamycin nucleus, and esters and ethers of a hydroxyl group at the27-position (following chemical reduction of the 27-ketone). Specificexamples of oximes, hydrazones, and hydroxylamines are of a ketone atthe 42-position (following oxidation of the 42-hydroxyl group) and of27-ketone of the rapamycin nucleus.

Examples of 42- and/or 31-esters and ethers of rapamycin are disclosedin the following patents, which are hereby incorporated by reference intheir entireties: alkyl esters (U.S. Pat. No. 4,316,885); aminoalkylesters (U.S. Pat. No. 4,650,803); fluorinated esters (U.S. Pat. No.5,100,883); amide esters (U.S. Pat. No. 5,118,677); carbamate esters(U.S. Pat. No. 5,118,678); silyl ethers (U.S. Pat. No. 5,120,842);aminoesters (U.S. Pat. No. 5,130,307); acetals (U.S. Pat. No. 551,413);aminodiesters (U.S. Pat. No. 5,162,333); sulfonate and sulfate esters(U.S. Pat. No. 5,177,203); esters (U.S. Pat. No. 5,221,670);alkoxyesters (U.S. Pat. No. 5,233,036); O-aryl, -alkyl, -alkenyl, and-alkynyl ethers (U.S. Pat. No. 5,258,389); carbonate esters (U.S. Pat.No. 5,260,300); arylcarbonyl and alkoxycarbonyl carbamates (U.S. Pat.No. 5,262,423); carbamates (U.S. Pat. No. 5,302,584); hydroxyesters(U.S. Pat. No. 5,362,718); hindered esters (U.S. Pat. No. 5,385,908);heterocyclic esters (U.S. Pat. No. 5,385,909); gem-disubstituted esters(U.S. Pat. No. 5,385,910); amino alkanoic esters (U.S. Pat. No.5,389,639); phosphorylcarbamate esters (U.S. Pat. No. 5,391,730);carbamate esters (U.S. Pat. No. 5,411,967); carbamate esters (U.S. Pat.No. 5,434,260); amidino carbamate esters (U.S. Pat. No. 5,463,048);carbamate esters (U.S. Pat. No. 5,480,988); carbamate esters (U.S. Pat.No. 5,480,989); carbamate esters (U.S. Pat. No. 5,489,680); hinderedN-oxide esters (U.S. Pat. No. 5,491,231); biotin esters (U.S. Pat. No.5,504,091); O-alkyl ethers (U.S. Pat. No. 5,665,772); and PEG esters ofrapamycin (U.S. Pat. No. 5,780,462).

Examples of 27-esters and ethers of rapamycin are disclosed in U.S. Pat.No. 5,256,790, which is hereby incorporated by reference in itsentirety.

Examples of oximes, hydrazones, and hydroxylamines of rapamycin aredisclosed in U.S. Pat. Nos. 5,373,014, 5,378,836, 5,023,264, and5,563,145, which are hereby incorporated by reference. The preparationof these oximes, hydrazones, and hydroxylamines is disclosed in theabove listed patents. The preparation of 42-oxorapamycin is disclosed inU.S. Pat. No. 5,023,263, which is hereby incorporated by reference.

Other compounds within the scope of “rapamycin derivatives” includethose compounds and classes of compounds referred to as “rapalogs” in,for example, WO 98/02441 and references cited therein, and “epirapalogs”in, for example, WO 01/14387 and references cited therein, thedisclosures of which are incorporated herein by reference in theirentireties.

Another compound within the scope of “rapamycin derivatives” iseverolimus, a 4-O-(2-hydroxyethyl)-rapamycin derived from a macrolideantibiotic produced by Streptomyces hygroscopicus (Novartis). Everolimusis also known as Certican, RAD-001 and SDZ-RAD.

Another preferred mTOR inhibitor is tacrolimus, a macrolide lactoneimmunosuppressant isolated from the soil fungus Streptomycestsukubaensis, Tacrolimus is also known as FK 506, FR 900506, Fujimycin,L 679934, Tsukubaenolide, Protopic and Prograf.

Another preferred mTOR inhibitor is ABT-578 an antiproliferative agent(Abbott Laboratories). ABT-578 is believed to inhibit smooth muscle cellproliferation with a cytostatic effect resulting from the inhibition ofmTOR.

Other preferred mTOR inhibitors include AP-23675, AP-23573, and AP-23841(Ariad).

Preferred rapamycin derivatives include everolimus, CCI-779 [rapamycin42-ester with 3-hydroxy-2-(hydroxymethyl)-2-methylpropionic acid; U.S.Pat. No. 5,362,718]; 7-epi-rapamycin; 7-thiomethyl-rapamycin;7-epi-trimethoxyphenyl-rapamycin; 7-epi-thiomethyl-rapamycin;7-demethoxy-rapamycin; 32-demethoxy-rapamycin; 2-desmethyl-rapamycin;and 42-O-(2-hydroxy)ethyl rapamycin (U.S. Pat. No. 5,665,772).

By way of a non-limiting example, Rapamycin is contacted with thedesired cells prior to, simultaneously with, and/or subsequent toexposing the cells to conditions that initiate iTreg conversion. Forexample, Rapa is contacted with the desired cells prior to,simultaneously with, and/or subsequent to exposing non-Treg cells toamino acid depletion conditions such as trypt depletion conditions. Insome instances, Rapa is contacted with the desired cells prior to,simultaneously with, and/or subsequent to exposing non-Treg cells toamino acid depletion conditions such as trypt depletion/catabolitesand/or a demethylating agent whereby the amino acid depletion conditionsand/or demethylating agent initiates iTreg conversion. In otherinstances, Rapa is contacted with the desired cells prior to,simultaneously with, and/or subsequent to exposing non-Treg cells to acombination of a demethylating agent and a HDACi.

Rapamycin may be used at a concentration of from about 0.01 μM to about10 μM, such as about 0.5 μM to about 2 μM, or about 1 μM.

It is also believed that the mTOR pathway is particularly sensitive tothe levels of nutrients, such as amino acids. Therefore, without wishingto be bound by any particular theory, it is believed that exposingnon-Tregs to an amino acid starvation environment such as Cryptdepletion/catabolites (e.g., low trypt/KYN) contributes to theregulation of the mTOR pathway.

Imprinting Stage:

An emerging paradigm in understanding the development of stable cellularlineages emphasizes the role of epigenetic mechanisms for the permanent,heritable fixation of distinct gene expression patterns. Molecularmechanisms of epigenetic imprinting include selective demethylation ofCpG motifs and histone modifications. It is believed that iTregconversion involves elements of epigenetic alterations such as DNAmethylation and histone modifications of at least the foxp3 locicorrelate with Foxp3. The present invention is partly based on theobservation that the selective association of chromatin remodelling witha stable Treg phenotype establishes a role of epigenetic imprinting inthe establishment of a committed regulatory T cell type.

Histone deacetylases (HDACs) regulate chromatin remodeling and geneexpression as well as the functions of a number of transcription factorsand nonhistone proteins. The results presented herein demonstrate that ahistone deacetylase inhibitor (HDACi) allows for the beneficialenhancement of converting non-Tregs into Tregs.

It has been shown that HDACi treatment enhances the production of Tregcells by either increasing thymic output of Treg cells or peripheralconversion of conventional T cells (non-Treg cells) into Treg cells.HDAC is are known to increase histone acetylation, resulting inchromatin remodelling and modulation of gene transcription. HDACitherapy also increases expression of Treg-associated gene such as Foxp3.

Suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA) targetclass I and II HDACs, respectively. HDAC7 is affected by vorinostat andHDAC9 by TSA; both class IIa HDACs have been linked to FoxP3 regulation(Tao et al., 2007, Nat Med 13:1299-307; Dokmanovic et al., 2007, MolCancer Ther 6:2525-34). In a preferred embodiment, an HDACi is used inconjunction with a demethylation agent to generate iTregs. An example ofa demethylating agent is 5-aza-2′-deoxycitidine (decitibine) or5-Azacytidine. Without wishing to be bound by any particular theory, itis believed that histone acetylation followed by demethylation isadvantageous for iTreg generation. For example, non-Tregs can beincubated with a combination of TSA/decitibine or SAHA/decitibinetreatment for iTreg conversion.

Therefore, the invention includes a method of exposing non-Tregs to anycombination of an amino acid depletion condition, a tryptophandepletion/catabolite, a demethylating agent, a DNA methyltransferaseinhibitor, an HDACi, and an inhibitor of mTOR (e.g., rapamycin), TGF-βto convert the non-Tregs into Tregs.

In some instances, the invention provides a combinational approach toconvert non-Tregs into Tregs or otherwise generate iTregs. Thecombinatorial approach includes an initial conversion stage, anoutgrowth stage that favors proliferation of Tregs, and an imprintingstage. In one embodiment, generation of iTregs involves exposingnon-Tregs to an amino acid starvation environment, including but is notlimited to a trypt depletion/catabolite condition (e.g., Low tryp/KYN),a demethylating agent (e.g., decitibine), or a combination of ademethylating agent with a HDACi to initiate the iTreg conversionprocess. To promote outgrowth of the converted Tregs, rapa or a mTORinhibitor can be added to the cells to favor Treg conversion andoutgrowth. To promote imprinting of iTregs, a demethylating agent and/orHDACi can be added to the cell culture. In some instances, the iTregsare imprinted after conversion has been initiated,

Expansion:

The invention includes converting non-Tregs or mixed populations ofTregs and non-Tregs into Tregs in the presence of a bead- or cell-basedartificial antigen-presenting cell system. Cells can be expanded using abead- or cell-based artificial antigen-presenting cell system.Regardless of the system used for cellular expansion, the cells can beexpanded prior to, simultaneously with, and/or subsequent to iTregconversion. For example, the cells can be expanded using a bead- orcell-based artificial antigen-presenting cell system before the initialiTreg conversion stage. Alternatively, the cells can be expanded using abead- or cell-based artificial antigen-presenting cell system after theinitial iTreg conversion stage but before the selective outgrowth stagethat favors proliferation of Tregs. Alternatively, the cells can beexpanded using a bead- or cell-based artificial antigen-presenting cellsystem after the outgrowth stage but before the imprinting stage.Alternatively, the cells can be expanded using a bead- or cell-basedartificial antigen-presenting cell system after the imprinting state.

Special cell-sized beads (e.g., magnetic iron-dextran beads) are usedthat are coated with antibodies to CD3 and CD28. The anti-CD28 providescritical signals for augmented activation and growth of thehypo-proliferative Treg cells. The use of anti-CD3/anti-CD28 beadsinduced robust proliferation of suppressor cells, having the effect ofplastic-bound antibody. CD28 stimulation also enhances the activation ofTreg cells and a present embodiment shows that beads coated withanti-CD3 and anti-CD28 (anti-CD3/CD28) antibody mixed with Treg cells atvarious ratios. As a non-limiting example, a 3:1 bead:T cell ratioexpands and preserves Treg function at a desirable level. CD28 is adisulfide bonded homodimer, expressed on the surface of the majority ofT cells (June et al., Immunol Today 11:211 (1990)). CD28 can beidentified by a number of commercially available CD28 monoclonalantibodies, as would be known to one of skill in the art. The ratios ofantibodies to CD3 and CD28 can be adjusted for optimal results. Thebeads can easily be removed by passing the cultured cells through amagnetic column. As an added advantage, the culture-expanded iTregsretain potent functional suppressor activity.

The present invention includes converting non-Tregs and mixedpopulations of non-Tregs and Tregs into Tregs in a cell-based artificialantigen presenting cell (aAPC) expansion system. In a non-limitingexample, cell-based aAPCs were created by electroporation of K562 cellswith CD32 and 4-1BBL expression plasmids. Using a combination of drugselection, cell sorting, and limiting dilution, high-expressing cloneswere isolated (Maus et al., 2002, Nature Biotechnol. 20:143-148).

A cell-based aAPC was designed to enable genetic manipulation of theexpression of different costimulatory molecules for the long term growthof T cells. The culture system was based on the fact that costimulatorysignals, in addition to those provided by CD28, are required for optimalCD8 cell growth. The human erythromyeloid CML cell line K562 (Lozzio etal., 1975, Blood 45:321-334) was used as a scaffold for the cellularaAPCs, because this cell line does not express HLA proteins that wouldpromote allogeneic responses. However, K562 do express ICAM (CD54) andLFA-3 (CD58), both of which promote interactions with T cell. Otheradvantages of using K562 cell include, but are not limited to, the factthat irradiated K562 cells can be introduced in the clinical setting asthese cells are mycoplasma-free, propagate in serum-free medium, and areeasily killed by natural killer (NK) cells.

The aAPC is engineered to have on its surface at least one moleculecapable of binding to a T-lymphocyte and inducing a primary activationevent and/or a proliferative response or capable of binding a moleculehaving such an affect thereby acting as a scaffold. For example, theaAPC is engineered to express a molecule that binds to the Fc portion ofan antibody. In some instances, the aAPC is engineered to stably expressa molecule capable of binding to the Fc portion of an antibody. The aAPCcan then be loaded or otherwise coated with or have attached thereto anyvariety of antibodies that recognize cell surface molecules present onthe surface of T lymphocytes, e.g., CD3, or a component of the TCR/CD3complex, CD28, 4-1BB, TCR, etc. Alternatively, the aAPC can be generatedby directly engineering a cell line to stably express the ligands forcell surface molecules present on the surface of T lymphocytes, e.g.CD3, or a component of the TCR/CD3 complex, CD28, 4-1BB, TCR, etc. TheaAPC can be further engineered to stably express one or moreco-stimulatory molecules, for example CD86 or 4-1BB ligand. In anon-limited example, an aAPC is engineered to express the humanlow-affinity Fcγ receptor, CD32 and the CD86 molecule. In anotherillustrative example, the aAPC is engineered to express CD32 and the4-1BB ligand. In one embodiment, the aAPC can be generated to expressmembrane bound ScFv or a fragment thereof, that recognize any cellsurface molecule of interest, such as CD3, CD28, 41BB and the like, orthat recognize other antibodies, such as through binding to the Fcportion. In this regard, the aAPC can be armed with secondary antibodiesthat bind through recognition of the Fc portion. The skilled artisanwould readily recognize that any variety and combination of stimulatoryand/or co-stimulatory molecules can be used in the context of thepresent invention.

The skilled artisan would appreciate, based upon the disclosure providedherein, that numerous immunoregulatory molecules can be used to producean almost limitless variety of aAPCs. For example, a primary signal,usually mediated via the T cell receptor/CD3 complex on a T cell,initiates the T cell activation process. Additionally, numerousco-stimulatory molecules present on the surface of a T cell are involvedin regulating the transition from resting T cell to cell proliferation.Such co-stimulatory molecules, also referred to as “co-stimulators”,which specifically bind with their respective ligands, include, but arenot limited to, CD28 (which binds with B7-1 [CD80], B7-2 [CD86]), PD-1(which binds with ligands PD-L1 and PD-L2), B7-H3, 4-1BB (binds theligand 4-1BBL), OX40 (binds ligand OX40L), ICOS (binds ligand ICOS-L),and LFA (binds the ligand ICAM). Thus, the primary stimulatory signalmediates T cell stimulation, but the co-stimulatory signal is thenrequired for T cell activation, as demonstrated by proliferation.

K562 cells can be transduced, either serially and/or in parallel, with awide plethora of exogenous nucleic acids to express a number ofmolecules thereby obtaining a library of aAPCs with desired phenotypes.With regard to use of K562 to produce aAPCs, the disclosures of U.S.patent application Ser. No. 10/336,135 (now published as U.S. PatentApplication Publication No, US2003/0147869A1) and International PatentApplication No. PCT/US03/00339 (now published as InternationalPublication No. WO 03/057171A2) are incorporated by reference as if setforth in their entirety herein.

The culture-expanded iTregs of the present invention are capable ofsuppressing an MLR, either with fresh CD4⁺ cells or cultured CD4⁺CD25⁻cells as responding T cells. In one embodiment the converted andexpanded iTregs inhibit the autologous proliferation of peripheral bloodcells. In another embodiment, the converted and expanded iTregs block orprevent GVHD, or inhibit or reverse the disease if already in progress.In yet another embodiment, the converted and expanded cells areintroduced into a different host; whereas in yet another embodiment, theiTregs are established as a cell line for continuous therapeutic use.Preferably, the host is a human host and the culture-expanded iTregs arehuman, although animals, including animal models for human diseasestates, are also included in this invention and therapeutic treatmentsof such animals are contemplated herein.

Following iTreg conversion using the methods of the invention, Tregs andiTregs can be expanded under appropriate conditions for growth of theTregs cells. Growth is allowed to progress for a time period selectedaccording to the final number of T cells required and the rate ofexpansion of the cells. Passaging of the cells may be undertaken duringthis period. Such a time period is normally between 3 and 10 days butcan be as long as 14 to 20 days or even longer providing the viabilityand continued proliferation of the T cells is maintained.

Therapeutic Application

The invention includes a method of suppressing an immune response in amammal for the treatment or prevention of an autoimmune condition ortransplantation rejection. The ex vivo culture-converted andculture-expanded iTregs with or without natural Tregs may bereintroduced to the host or to another patient by a number ofapproaches. Preferably, they are injected intravenously. Optionally, thehost may be treated with agents to promote the in vivo function andsurvival of the stimulated cells. Of course, the culture-expanded iTregsmay also be reintroduced in a variety of pharmaceutical formulations.These may contain such normally employed additives as binders, fillers,carriers, preservatives, stabilizing agents, emulsifiers, and buffers.Suitable diluents and excipients are, for example, water, saline, anddextrose, as utilized in the methods described below.

This method thus provides a method of achieving an immunosuppressiveeffect in a mammal, i.e. a method of preventing an immune response. Thecondition or disease typified by an aberrant immune response may be anautoimmune disease, for example diabetes, multiple sclerosis, myastheniagravia, neuritis, lupus, rheumatoid arthritis, psoriasis or inflammatorybowel disease. Conditions in which immune suppression would beadvantageous include conditions in which a normal or an activated immuneresponse is disadvantageous to the mammal, e.g. allotransplantation ofe.g. body fluids or parts, to avoid rejection, or in fertilitytreatments in which inappropriate immune responses have been implicatedin failure to conceive and miscarriage. The use of such cells before,during, or after transplantation avoids extensive chronic graft versushost disease which may occur in patients being treated (e.g. cancerpatients). The cells may be converted immediately after harvest orstored (e.g. by freezing) prior to expansion or after expansion andprior to their therapeutic use. The therapies may be conducted inconjunction with known immunosuppressive therapies.

The methods of the present invention are particularly useful for humans,but may also be practiced on veterinary subjects. An “individual,”“subject,” “patient” or “host” referred to herein is a vertebrate,preferably a mammal. More preferably, such individual is a human and theculture-expanded cells are human, although animals, including animalmodels for human disease states, are also included in this invention andtherapeutic treatments of such animals are contemplated herein. Suchanimal models can be used to test and adjust the compositions andmethods of this invention, if desired. Certain models involve injectingin-bred animals with established cell populations. Also useful arechimeric animal models, described in U.S. Pat. Nos. 5,663,481, 5,602,305and 5,476,993; EP application 379,554; and International Appl. WO91/01760. Non-human mammals include, but are not limited to, veterinaryor farm animals, sport animals, and pets. Accordingly, as opposed toanimal models, such animals may be undergoing selected therapeutictreatments.

The present invention encompasses a method of reducing and/oreliminating an immune response to a transplant in a recipient byadministering to the recipient of the transplant an amount of iTregseffective to reduce or inhibit host rejection of the transplant. Withoutwishing to be bound to any particular theory, the iTregs that areadministered to the recipient of the transplant inhibit the activationand proliferation of the recipient's T cells or induce tolerance.

The transplant can include a biocompatible lattice or a donor tissue,organ or cell, to be transplanted. An example of a transplant mayinclude but is not limited to skin cells or tissue, bone marrow, andsolid organs such as heart, pancreas, kidney, lung and liver. In someinstances, the transplant is a nucleic acid or a protein.

Based upon the disclosure provided herein, iTregs can be obtained fromany source, for example, from the tissue donor, the transplant recipientor an otherwise unrelated source (a different individual or speciesaltogether). The iTregs may be autologous with respect to the T cells(obtained from the same host) or allogeneic with respect to the T cells.In the case where the iTregs are allogeneic, the iTregs may beautologous with respect to the transplant to which the T cells areresponding to, or the iTregs may be obtained from a mammal that isallogeneic with respect to both the source of the T cells and the sourceof the transplant to which the T cells are responding to. In addition,the iTregs may be xenogeneic to the T cells (obtained from an animal ofa different species), for example rat iTregs may be used to suppressactivation and proliferation of human T cells.

Another embodiment of the present invention encompasses the route ofadministering iTregs to the recipient of the transplant. iTregs can beadministered by a route which is suitable for the placement of thetransplant, i.e. a biocompatible lattice or a donor tissue, organ orcell, nucleic acid or protein, to be transplanted. iTregs can beadministered systemically, i.e., parenterally, by intravenous injectionor can be targeted to a particular tissue or organ, such as bone marrow.iTregs can be administered via a subcutaneous implantation of cells orby injection of the cells into connective tissue, for example, muscle.

Tregs can be suspended in an appropriate diluent, at a concentration ofabout 5×10⁶ cells/ml. Suitable excipients for injection solutions arethose that are biologically and physiologically compatible with theTregs and with the recipient, such as buffered saline solution or othersuitable excipients. The composition for administration can beformulated, produced and stored according to standard methods complyingwith proper sterility and stability.

The dosage of the Tregs varies within wide limits and may be adjusted tothe mammal requirements in each particular case. The number of cellsused depends on the weight and condition of the recipient, the numberand/or frequency of administrations, and other variables known to thoseof skill in the art.

Between about 10⁵ and about 10¹³ Tregs per 100 kg body weight can beadministered to the mammal. In some embodiments, between about 1.5×10⁶and about 1.5×10¹² cells are administered per 100 kg body weight. Insome embodiments, between about 1×10⁹ and about 5×10¹¹ cells areadministered per 100 kg body weight. In some embodiments, between about4×10⁹ and about 2×10¹¹ cells are administered per 100 kg body weight. Insome embodiments, between about 5×10⁸ cells and about 1×10¹⁰ cells areadministered per 100 kg body weight.

In another embodiment of the present invention, Tregs are administeredto the recipient prior to, or contemporaneously with a transplant toreduce and/or eliminate host rejection of the transplant. While notwishing to be bound to any particular theory, Tregs can be used tocondition a recipient's immune system to the transplant by administeringTregs to the recipient, prior to, or at the same time as transplantationof the transplant, in an amount effective to reduce, inhibit oreliminate an immune response against the transplant by the recipient's Tcells. The Tregs affect the T cells of the recipient such that the Tcell response is reduced, inhibited or eliminated when presented withthe transplant. Thus, host rejection of the transplant may be avoided,or the severity thereof reduced, by administering Tregs to therecipient, prior to, or at the same time as transplantation.

In yet another embodiment, Tregs can be administered to the recipient ofthe transplant after the administration of the transplant. Further, thepresent invention comprises a method of treating a patient who isundergoing an adverse immune response to a transplant by administeringTregs to the patient in an amount effective to reduce, inhibit oreliminate the immune response to the transplant, also known as hostrejection of the transplant.

Therapy to Inhibit Adverse Immune Responses Following Transplantation

The present invention includes a method of using Tregs as a therapy toinhibit graft versus host disease or graft rejection followingtransplantation. Accordingly, the present invention encompasses a methodof contacting a donor transplant, for example a biocompatible lattice ora donor tissue, organ or cell, with Tregs prior to transplantation ofthe transplant into a recipient. The Tregs serve to ameliorate, inhibitor reduce an adverse response by the donor transplant against therecipient.

As discussed elsewhere herein, Tregs can be obtained from any source,for example, from the tissue donor, the transplant recipient or anotherwise unrelated source (a different individual or speciesaltogether) for the use of eliminating or reducing an unwanted immuneresponse by a transplant against a recipient of the transplant.Accordingly, Tregs can be autologous, allogeneic or xenogeneic to thetissue donor, the transplant recipient or an otherwise unrelated source.

In an embodiment of the present invention, the transplant is exposed toTregs prior, at the same time, or after transplantation of thetransplant into the recipient. In this situation, an immune responseagainst the transplant caused by any alloreactive recipient cells wouldbe suppressed by the Tregs present in the transplant. The Tregs areallogeneic to the recipient and may be derived from the donor or from asource other than the donor or recipient. In some cases, Tregsautologous to the recipient may be used to suppress an immune responseagainst the transplant. In another case, the Tregs may be xenogeneic tothe recipient, for example mouse or rat Tregs can be used to suppress animmune response in a human. However, it is preferable to use human Tregsin the present invention.

In another embodiment of the present invention, the donor transplant canbe “preconditioned” or “pretreated” by treating the transplant prior totransplantation into the recipient in order to reduce the immunogenicityof the transplant against the recipient, thereby reducing and/orpreventing graft versus host disease or graft rejection. The transplantcan be contacted with cells or a tissue from the recipient prior totransplantation in order to activate T cells that may be associated withthe transplant. Following the treatment of the transplant with cells ora tissue from the recipient, the cells or tissue may be removed from thetransplant. The treated transplant is then further contacted with Tregsin order to reduce, inhibit or eliminate the activity of the T cellsthat were activated by the treatment of the cells or tissue from therecipient. Following this treatment of the transplant with Tregs, theTregs may be removed from the transplant prior to transplantation intothe recipient. However, some Tregs may adhere to the transplant, andtherefore, may be introduced to the recipient with the transplant. Inthis situation, the Tregs introduced into the recipient can suppress animmune response against the recipient caused by any cell associated withthe transplant. Without wishing to be bound to any particular theory,the treatment of the transplant with Tregs prior to transplantation ofthe transplant into the recipient serves to reduce, inhibit or eliminatethe activity of the activated T cells, thereby preventing restimulation,or inducing hyporesponsiveness of the T cells to subsequent antigenicstimulation from a tissue and/or cells from the recipient. One skilledin the art would understand based upon the present disclosure, thatpreconditioning or pretreatment of the transplant prior totransplantation may reduce or eliminate the graft versus host response.

For example, in the context of umbilical cord blood, bone marrow orperipheral blood stem cell (hematopoietic stem cell) transplantation,attack of the host by the graft can be reduced, inhibited or eliminatedby preconditioning the donor marrow by using the pretreatment methodsdisclosed herein in order to reduce the immunogenicity of the graftagainst the recipient. As described elsewhere herein, a donorhematopoietic stem and progenitor cell source can be pretreated withTregs from any source, preferably with recipient Tregs in vitro prior tothe transplantation of the donor marrow into the recipient. In apreferred embodiment, the donor marrow is first exposed to recipienttissue or cells and then treated with Tregs. Although not wishing to bebound to any particular theory, it is believed that the initial contactof the donor hematopoietic stein and progenitor cell source withrecipient tissue or cells function to activate the T cells in the donormarrow. Treatment of the donor marrow with the Tregs induceshyporesponsiveness or prevents restimulation of T cells to subsequentantigenic stimulation, thereby reducing, inhibiting or eliminating anadverse affect induced by the donor marrow on the recipient.

In an embodiment of the present invention, a transplant recipientsuffering from graft versus host disease or graft rejection may betreated by administering Tregs to the recipient to reduce, inhibit oreliminate the severity thereof from the graft versus host disease wherethe Tregs are administered in an amount effective to reduce or eliminategraft versus host disease.

In this embodiment of the invention, preferably, the recipient's Tregsmay be obtained from the recipient prior to the transplantation and maybe stored and/or expanded in culture to provide a reserve of Tregs insufficient amounts for treating an ongoing graft versus host reaction.However, as discussed elsewhere herein, Tregs can be obtained from anysource, for example, from the tissue donor, the transplant recipient oran otherwise unrelated source (a different individual or speciesaltogether),

Advantages of Using Tregs

Based upon the disclosure herein, it is envisioned that the Tregs of thepresent invention can be used in conjunction with current modes, forexample the use of immunosuppressive drug therapy, for the treatment ofhost rejection to the donor tissue or graft versus host disease. Anadvantage of using Tregs in conjunction with immunosuppressive drugs intransplantation is that by using the methods of the present invention toameliorate the severity of the immune response in a transplantrecipient, the amount of immunosuppressive drug therapy used and/or thefrequency of administration of immunosuppressive drug therapy can bereduced. A benefit of reducing the use of immunosuppressive drug therapyis the alleviation of general immune suppression and unwanted sideeffects associated with immunosuppressive drug therapy.

It is also contemplated that the cells of the present invention may beadministered into a recipient as a “one-time” therapy for the preventionor treatment of host rejection of donor tissue or graft versus hostdisease. A one-time administration of Tregs into the recipient of thetransplant eliminates the need for chronic immunosuppressive drugtherapy. However, if desired, multiple administrations of Tregs may alsobe employed.

The invention described herein also encompasses a method of preventingor treating transplant rejection and/or graft versus host disease byadministering Tregs in a prophylactic or therapeutically effectiveamount for the prevention, treatment or amelioration of host rejectionof the transplant and/or graft versus host disease. Based upon thepresent disclosure, a therapeutic effective amount of Tregs is an amountthat inhibits or decreases the number of activated T cells, whencompared with the number of activated T cells in the absence of theadministration of Tregs. In the situation of host rejection of thetransplant, an effective amount of Tregs is an amount that inhibits ordecreases the number of activated T cells in the recipient of thetransplant when compared with the number of activated T cells in therecipient prior to administration of the Tregs. In the case of graftversus host disease, an effective amount of Tregs is an amount thatinhibits or decreases the number of activated T cells present in thetransplant.

An effective amount of Tregs can be determined by comparing the numberof activated T cells in a recipient or in a transplant prior to theadministration of Tregs thereto, with the number of activated T cellspresent in the recipient or transplant following the administration ofTregs thereto. A decrease, or the absence of an increase, in the numberof activated T cells in the recipient of the transplant or in thetransplant itself that is associated with the administration of Tregsthereto, indicates that the number of Tregs administered is atherapeutic effective amount of Tregs.

The invention also includes methods of using Tregs of the presentinvention in conjunction with current mode, for example the use ofimmunosuppressive drug therapy, for the treatment of host rejection tothe donor tissue or graft versus host disease. An advantage of usingTregs in conjunction with immunosuppressive drugs in transplantation isthat by using the methods of the present invention to ameliorate theseverity of the immune response following transplantation, the amount ofimmunosuppressive drug therapy used and/or the frequency ofadministration of immunosuppressive drug therapy can be reduced. Abenefit of reducing the use of immunosuppressive drug therapy is thealleviation of general immune suppression and unwanted side effectsassociated with immunosuppressive drug therapy.

It should be understood that the methods described herein may be carriedout in a number of ways and with various modifications and permutationsthereof that are well known in the art. It may also be appreciated thatany theories set forth as to modes of action or interactions betweencell types should not be construed as limiting this invention in anymanner, but are presented such that the methods of the invention can bemore fully understood.

The following examples further illustrate aspects of the presentinvention. However, they are in no way a limitation of the teachings ordisclosure of the present invention as set forth herein.

These methods described herein are by no means all-inclusive, andfurther methods to suit the specific application will be apparent to theordinary skilled artisan. Moreover, the effective amount of thecompositions can be further approximated through analogy to compoundsknown to exert the desired effect.

EXAMPLES

The invention is now described with reference to the following Examples.These Examples are provided for the purpose of illustration only, andthe invention is not limited to these Examples, but rather encompassesall variations which are evident as a result of the teachings providedherein.

The experiments disclosed herein were conducted to develop and optimizeapproaches for the conversion and large-scale expansion of human CD4⁺25⁺T cells from CD4⁺ or CD4⁺25⁻ T cells, inducible Tregs (iTregs), forclinical trials including but not limited to prevent graft-versus-hostdisease (GVHD) after allogeneic transplant. The results disclosed hereindemonstrate that iTregs can be generated using a combinatorial approachusing tryptophan depletion/catabolites or a demethylating agent toinitiate the iTreg conversion process, an mTOR inhibitor (e.g., rapa) tofavor Treg conversion and outgrowth, and the later addition of ademethylating agent or a histone deacetylation inhibitor (HDACi) forimprinting iTregs after conversion.

Example 1 Expansion of CD4⁺25⁺Tregs

It has been previously shown that using research-grade beads forextensive negative selection followed by positive selection for CD4⁺25⁺Tregs, and expanding the Tregs with anti-CD3/28 microbeads+IL-2 failedto generate uniformly suppressive cells. In addition, isolation ofCD4⁺127lo cells (CD127lo cells are approximately 10% of CD4⁺ T cells)followed by anti-CD3/28 microbead/IL2 expansion did not permit uniformsuppression (FIG. 1B). Although anti-CD3 mAb loaded K562 cells modifiedto express an FoR (CD64) and CD86 (KT64/86) was superior to anti-CD3/28beads for expanding cells, a high level of suppression was not uniformlyseen with either approach (FIG. 1B).

Adding rapa, which is an mTOR inhibitor, (labeled as +) reduced meanexpansion rates by 30-fold with beads resulting in≦10-fold meanexpansion rates by day 14. Rapa added to KT64/86 driven cultures (FIG.1A) reduced mean expansion by 10-fold and improved suppression, althoughin some instances suppression was modest at high ratios (1:4) of Tregsto PBMNCs. The addition of TGFβ (10 ng/ml) to the cells did not increasesuppression.

Since approximately 1-2% of CD4⁺ T cells are CD25bright natural Tregs(nTregs) and incubation of these cells with rapa reduces theirexpansion, it is believed that at best, incubating nTregs with rapa isinefficient and at worst, the variability in suppressor cell potency hasprecluded clinical trials of ex vivo expanded peripheral blood Tregs todate. The next set of experiments were designed to convert CD4⁺ T cellsinto inducible Tregs (iTregs) in order to utilize the 98-99% of CD4⁺ Tcells that are discarded when isolating CD4⁺ T cells that are CD25brightnTregs. Without wishing to be bound by any particular theory, it isbelieved that converting CD4⁺ T cells into iTregs also reduce thelikelihood that non-Treg contaminants would result in loss ofsuppression.

Example 2 Culturing CD4⁺25⁻ T Cells and iTreg Generation

CD4⁺25⁻ T cells, when cultured with allogeneic TLR9 activated allogeneicplasmacytoid dendritic cells (pDCs), but not B-cells, lead to thegeneration of iTregs, CD4⁺25⁺FoxP3⁺ suppressor cells (FIG. 2). It wasalso observed that iTregs (closed), but not T cells primed to TLRactivated B cells (open squares), were potently suppressive of a naïveMLR culture (x-axis are x 104; MLR used 105 naïve T cells; FIG. 3). Itwas observed that iTreg generation was dependent upon the enzymeindoleamine 2,3-dioxygenase (IDO) since IDO inhibition by 1-methyl-trypt(1MT) prevented conversion (FIG. 3B). Since IDO catabolizes tryptophaninto kynurenines (KYN), it is believed that iTregs can be generated in Bcell cultures using KYN. As shown in FIG. 3C, KYN resulted in potent MLRsuppression that was not blocked by the addition of 1MT (KYN isdownstream from IDO).

While these data provide proof-of-principle that KYN supports iTreggeneration, the number of output iTregs equaled the input CD4⁺25⁻ Tcells during MLR culture, To optimize iTreg conversion and expansion,experiments were designed to switch from the culture system to using abead or cell based artificial APCs system and to compare the level ofiTreg generation when CD4⁺25⁻ T cells were exposured to low levels oftrypt/KYN or decitibine. To ensure naïve T cell conversion, rather thannTreg or T effector cell expansion, CD4⁺25⁻45RA⁺ T cells were used.

For initial studies, to ensure inducible Treg (iTreg) conversion wasbeing examined and not natural Treg (nTreg) expansion, CD4⁺25⁻45RA⁺ Tcells are isolated from ficolled buffy coat cells by negative selection(CD8, CD11b, CD16, CD19, CD36, CD56 CD25) followed by CD45RA positiveselection using beads. Cells are cultured in X-Vivo-15 or trypt-freemedia, as indicated, along with human ABneg serum (10%) and L-glutamine.rIL-2 (300 U/ml: Chiron) is added on day 2 and with re-feeds.

In order to assay for the presence of non-Tregs and nTregs, multi-colorphenotyping can be employed pre- and post-isolation and on days 7, 10,14, 21 for both non-Tregs and nTregs (CD4/25/127lo/FoxP3) forco-expression of adhesion molecules (e.g., CD62L; LFA-1), chemokinereceptors (e.g., CCR4-9; CXCR3), and cytokines (IL2, IL17, TNFa, IL10),Phosphorylated-Stat5 and p-Akt can be analyzed at 15′ post-stimulationwith PMA/ionomycin.

To simulate IDO effects, cells were cultured in low tryp+KYN using KYNat 10 μM+trypt at 1 μM until day 7 then 5 μM trypt beyond day 7 tosupport expansion after iTreg “imprinting” has occurred. Decitibine wasadded at 1-5 μM (final) to day 3 cultures. Cultures were expanded withanti-CD3/28 mAb-loaded, irradiated KT32 (FcR+) cells, Anti-CD3/28 beads(3 beads:1 cell) or 100 Gy irradiated, anti-CD3 (OKT3) loaded KT64/86cells (1 KT cell:2 CD4⁺25⁻ T cells) are added on day 0. Cell density ismaintained at 0.2-2×106/ml by 50% vol/vol re-feeds, By 10 days, diluentcultured cells expanded about 30-fold, decibine about 11 fold and lowtryp/KYN about 5-fold; CD4+CD127loFoxP3hi cells were 1%, 16%, and 13%,respectively (FIG. 4; upper, FoxP3hi; lower box, FoxP3int,lo). FoxP3levels are related to suppressor potency and stability (Wan et al.,2007, Nature 445:766-70; Pillai et al., 2007, Clin Immunol 123:18-29;Williams et al., 2007, Nat Immunol 8:277-84). iTreg suppression of CD8+T cell proliferation (1:16 ratio), assessed by quantifying CFSEdye-dilution, was 18%, 41% and 76%, respectively, suggesting thatFoxP3int cells present in the low trypt/KYN group were contributing tosuppressor cell potency. Taken together, these data indicate that largeiTreg numbers could be generated without discarding 98-99% of CD4+ Tcells needed to obtain CD4+25br cells and with expansion by day 10. Instudies using umbilical cord blood (UCB) nTregs, day 11 culturesre-stimulated with KT cells expanded an additional 20-fold compared tono re-stimulation. These data indicate that iTregs can be generated inhigh numbers and suggest a strategy for maximizing iTreg expansion.

Example 3 iTreg Generation Using Histone Acetylation Followed ByDemethylation

Recent studies have analyzed HDACi (suberoylanilide hydroxamic acid:SAHA; trichostatin A: TSA). SAHA and TSA target class I and II HDACs atμM and nM amounts, respectively (Dokmanovic, et al., 2007, Mol CancerRes 5:981-9; Dangond et al., 1998, Biochem Biophys Res Commun247:833-7). HDAC7 is affected by vorinostat and HDAC9 by TSA; both classIIa HDACs have been linked to FoxP3 regulation (Tao et al., 2007, NatMed 13:1299-307; Dokmanovic et al., 2007, Mol Cancer Ther 6:2525-34). Ithas been observed that SAHA at 100 μM on day 0 was toxic, whereas SAHAat 300 nM on day 3 did not inhibit T cell expansion but also did notresult in iTregs.

While HDACi is insufficient to result in iTregs in vitro (Tao et al.,2007, Nat Med 13:1299-307), it is believed that HDACi would be effectivewhen used with decitibine, due to the known increase in histonemethylation and deacetylation in Tregs and potential benefits ofepigenetic modification on FoxP3 and Treg stability. Neither raps, TGFb(10 ng/ml), SAHA (300 nM) (not shown) nor TSA (100 nM) (FIG. 5) inducedCD4+127loFoxP3hi cells by day 10. It was observed in cultures with day 3SAHA or TSA, decitibine added on day 7 markedly increasedCD4+127loFoxP3hi cells by day 10 from 4.6% (diluent) to 44.5%(SAHA/decitibine), indicating that histone acetylation followed bydemethylation was advantageous for iTreg generation (FIG. 5A). Day 10expansions were: diluent (20-fold), TSA (15-fold), TSA/decibine(6-fold), and SAHA/decitibine (7-fold). Although there was a decrementin total expansion rates between days 7-10 with decitibine, these cellswere observed to recover and undergo vigorous growth. Based upon thepercent FoxP3hi and FoxP3int cells in the SAHA/decitibine group, it isbelieved that suppression potency will exceed that observed with lowtryp/KYN cultures in FIG. 4.

In the same experiment, bead expanded cultures revealed the followingfor CD4⁺127loFoxP3hi vs FoxP3int/lo: diluent (1.7, 21.8%); TSA (1.6%,15.4%); TSA/decitibine (8.5%, 43.5%); SAHA/decitibine (7,9%; 41.1%).Despite the high iTreg levels, day 10 expansion was already 20-fold forTSA/decibine and 23-fold for SAHA/decitibine.

The next set of experiments were designed to compare the effects of aHDACi in a bead- or cell-based artificial antigen-presenting cell system(KT32) for generating iTregs (FIGS. 5B and 5C). Purified human naïve Tcells (CD4⁺, CD25⁻, CD45RA⁺) were stimulated with KT32 (K562 engineeredto express human CD32) loaded with anti-CD3 and CD28 antibodies. Thecells were cultured for 3 days and SAHA added a single time at theindicated concentrations, and analyzed 7 days later (day 10) for Foxp3expression (FIG. 5B). Purified human naïve T cells (CD4⁺, CD25⁻,CD45RA⁺) were stimulated with KT32 (K562 engineered to express humanCD32) loaded with anti-CD3 and CD28 antibodies. The cells were culturedfor 3 days and Trichostatin A added a single time at the indicatedconcentrations, and analyzed 7 days later (day 10) for Foxp3 expression.

The next set of experiments were designed to compare the effects of ademethylating agent in a bead- or cell-based artificialantigen-presenting cell system (KT32) for generating iTregs (FIGS. 5Dand 5E). Purified human naïve T cells (CD4⁺, CD25⁻, CD45RA⁺) werestimulated with clinical grade anti-CD3/28 beads or with KT32 (K562engineered to express human CD32) loaded with anti-CD3 and CD28antibodies. The cells were cultured for 3 days and Decitibine added asingle time at the indicated concentrations, and analyzed 7 days later(day 10) for Foxp3 expression (FIG. 5D). Purified human naïve T cells(CD4⁺, CD25⁻, CD45RA⁺) were stimulated with clinical grade anti-CD3/28beads or with KT32 (K562 engineered to express human CD32) loaded withanti-CD3 and CD28 antibodies. The cells were cultured for 3 days and5-Azacytidine added a single time at the indicated concentrations, andanalyzed 7 days later (day 10) for Foxp3 expression (FIG. 5E).

The next set of experiments were designed to compare the effects of thecombination of an HDACi with a demethylating agent in a bead- andcell-based antigen-presenting cell system (FIG. 5F). Purified humannaïve T cells (CD4⁺, CD25⁻, CD45RA⁺) were stimulated with clinical gradeanti-CD3/28 beads or with KT32 (K562 engineered to express human CD32)loaded with anti-CD3 and CD28 antibodies. On day 3 of culture, thespecified HDACi were added. On day 7, a single dose of Decitibine (1 μM)was added and cultures analyzed 3 days later (day 10) for Foxp3expression.

Example 4 iTreg Generation and Monitoring

Methylation and histone deacetylation typically suppress genetranscription (Tao et al., 2007, Curr Opin Immunol 19:589-95). Incomparison to non-Tregs, natural Tregs (nTregs) are hypomethylated andit has been observed that complete demethylation and histonemodifications occur in the FoxP3 locus of nTregs (Floess et al., 2007,PLoS Biol 5:e38; Baron et al., 2007, Eur J Immunol 37:2378-89).Consistent with importance of epigenetic modification for iTregstability, re-stimulation of TGFb-generated iTregs in the absence ofTGFb results in loss of FoxP3 and suppression (Floess et al., 2007, PLoSBiol 5:e38). In rodents, a histone deacetylation inhibitor (HDACi)supported in vivo rodent iTreg generation in nTreg-depleted but notTreg-replete recipients (Tao et al., 2007, Nat Med 13:1299-307). It isreported that nTregs are less dependent than non-Tregs on the mTOR/Aktpathway. Rapa, an mTOR inhibitor, has been shown to suppress non-Tregsand favor iTreg conversion (Zeiser et al., 2008, Blood 111:453-62;Battaglia et al., 2005, Blood 105:4743-8; Coenen et al., 2006, Blood107:1018-23; Haxhinasto et al., 2008, J Exp Med 205(3):565-74).

The following experiments were designed to determine the optimalapproach for iTreg generation and expansion, to quantify iTreg potencyand functional stability in vitro and in vivo, and to performlarge-scale GMP-production of iTregs for clinical trial implementation.

The results presented herein demonstrate that iTregs can be generated intryptophan (trypt) depletion conditions. In addition, the resultsdemonstrate the use of a combinatorial approach to generate iTregs usingtrypt depletion/catabolites or a demethylating agent to initiate theiTreg conversion process, rapa to favor Treg conversion and outgrowth,and the later addition of a demethylating agent or HDACi for imprintingiTregs. Potency and suppressor function stability of the iTregs can bemeasured in vitro and in a xenogeneic GVHD model.

A matrix-type approach may be used for sequential and when desired,experiments are designed for combinatorial testing of optimalconcentrations of single agents guided by assays described below.

Decitibine

Around 1-5 μM decitibine may be added day 3 vs days 3+7 (to furtherimprint cells). Hypomethylation is assessed using bisulfite sequencing(Baron et al., 2007, Eur J Immunol 37:2378-89; Issa et al., 2005, J ClinOncol 23:3948-56) and comparisons made to days 7 & 21 between expandednTregs and flow-sorted adult peripheral blood Tregs. H3 and H4 histoneacetylation can be assessed under various conditions by Western blot andELISA (O'Connor et al., 2006, J Clin Oncol 24:166-73; Garcia-Manero etal., 2008, Blood 111:1060-6) to determine the extent to which iTregprotocols are associated with histone acetylation and HDACi alter thispattern. CHOP staining may be used as an indicator of the GCN2 kinasestress response (for trypt/KYN) (Sharma et al., 2007, J Clin Invest117:2570-82), and p-serine473-Akt (for rap) (Crellin et al., 2007, Blood109:2014-22) may be monitored at time points as above. Without wishingto be bound by any particular theory, an acceptable range for a using ademethylating agent (e.g., 5-aza-2′-deoxycitidine (decitibine),5-Azaeytidine) according to the invention is believed to be around 0.1to 100 μM.

Trypt/KYN

A preferred approach to generate iTregs is to induce a stress response.For example addition of trypt at 1 μM for 7 days followed by morepermissive growth conditions, trypt at 5 μM; trypt metabolites (3HK,3HAA, QA, AA, L-KYN; Sigma) are added at 10 μM (final) on day 0 and withre-feeds. Without wishing to be bound by any particular theory, anacceptable range for using trypt or trypt metabolite according to theinvention is around 1-100 μM.

SAHA

SAHA (Merck) may be added at ranges of 300-1000 nM on day 7 and withre-feeding in cultures initiated with decitibine or low trypt/KYN.Conversely, decitibine may be added on day 7 to SAHA initiated cultures(see FIG. 5). Without wishing to be bound by any particular theory, anacceptable range for using a HDACi according to the invention is around10 nM to 1 μM for TSA, and around 50 nM to 5 μM for SAHA.

Rapa

Rapa (109 nM; Wyeth-Ayerst) may be added on 0 and with re-feeds alongwith decitibine, trypt/KYN or HDACi (day 3 or day 7). Alternatively,rapa may be added on day 7 and with re-feeds after initial iTregimprinting has occurred to facilitate iTreg conversion and preventnon-Treg outgrowth, minimizing the impact of day 0 rapa on cell yield.Without wishing to be bound by any particular theory, an acceptablerange for using Rapa according to the invention would be about 10-1000nM Rapa (Wyeth).

Example 5 In Vivo Potency of Expanded iTregs

To quantify in vivo GVHD inhibitory capacity, C57BL/6-IL2Rgc/ragknockout mice were macrophage depleted using clodronate, sublethallyirradiated, and given PBMNC (30×106)±a suboptimal number (10×106) ofanti-CD3/28 beads or irradiated, anti-CD3/28 mAb loaded KT cell lineexpanded UCB nTregs iv. While both nTreg sources significantly reducedGVHD lethality, nTregs generated with KT cells were superior to thosewith beads. Day 10 peripheral blood analysis indicated that KT vs. beadexpanded Tregs were present in significantly higher numbers. Human CD4+and CD8+ T cells present in the spleen at the time of death were reducedby nTregs, with the greatest effects observed using KT expanded nTregs.Human PBMNCs cause severe GVHD of the liver, lung and ileum (meanscores>3.0 on a 4 point scale), associated with human T cellinfiltration into these organs (not shown). This model is useful toquantify in vivo potency of expanded iTregs vs nTregs cultured and tofacilitate iTreg choice for trials.

In vitro potency of expanded iTregs vs nTregs may be assayed forsuppression in MLR and anti-CD3 mAb driven, CD8+ CFSE T cellproliferation assays. Those approaches that are the most potent via invitro assays while permitting at least a 10-fold expansion over CD4⁺25⁻input number are assessed by dose titrations of iTregs vs nTregs usingthe above described xenogeneic GVHD model. iTregs and nTregs aremismatched for PBMNCs at HLA-A2 or HLA-B7 to permit HLA flow typing ofperipheral blood on days 7-14 and tissue analysis along with survival,weight loss and GVHD scoring.

Stability of iTregs can be measured by assessing FoxP3. FoxP3 acts in adose-dependent non-binary fashion to control suppressor function (Wan etal., 2007, Nature 445:766-70). A transient Treg phenotype has beendescribed (Pillai et al., 2007, Clin Immunol 123:18-29). FoxP3 lossresults in suppression loss (Williams et al., 2007, Nat Immunol8:277-84). iTregs and nTregs may be phenotyped for TNFα, IFNγ, IL17 andreceptors that induce Th17 cells (IL6R, IL23R, CCR4, CCR6, RORgt) andexposed to conditions for Th17 generation and expansion (anti-CD3/28mAbs; IL-23, IL6, IL1b, TNFα, TGFβ1, and mAbs to IL4, IL12, and IFNγ(Weaver et al., 2006, Immunity 24:677-88; Nurieva et al., 2007, Nature448:480-3; Yang et al., 2008, Immunity 28:29-39; Stockinger et al.,2007, Curr Opin Immunol 19:281-6; Veldhoen et al., 2006, Immunity24:179-89; Korn et al., 2007, Nature 448:484-7; Singh et al., 2008, JImmunol 180:214-21). Experiments are designed to analyze Tregs for Th17,Th1 and Th2 conversion along with FoxP3 expression in the xenogeneicGVHD model with HLA typing of PB and GVHD organs. If Th17 cells areseen, all trans-retinoic acid (ATRA), 0.1-1 μM (Schambach et al., 2007,Eur J Immunol 37:2396-9; Elias et al., 2008, Blood 111:1013-20; Benson,et al., 2007, J Exp Med 204:1765-74), may be added on day 0 or on day 7,to minimize its anti-proliferative effects and repeat potency andstability studies.

Example 6 Clinical Trial

CD4⁺25⁺ natural Tregs (nTregs) have been shown to have the ability toprevent GVHD, donor bone marrow graft rejection, and in speeding immunerecovery in GVHD-prone mice (Gregori et al., 2005, Curr Opin Hematol12:451-6; Blazar et al., 2005, Biol Blood Marrow Transplant 11:46-9).Clinical testing adult peripheral blood (PB) Tregs has been hampered ofby the low frequency and less distinct CD4/25br subset and availabilityof GMP reagents for rigorous Treg purification (June et al., 2006, SeminImmunol 18:78-88). Isolation and expansion protocols can result insuppression loss when about 5% non-Treg are present. Umbilicord bloodTregs exist at higher frequency, have a distinct CD4/25br subset readilypurified using CD25 mAb-coated beads and can be expanded by about200-1000 fold in <3 weeks using anti-CD3/28 mAb-coated beads+IL-2(Porter et al., 2006, Transplantation 82:23-9; Godfrey et al., 2005,Blood 105:750-8).

It has been shown that CD4⁺25⁻ T-cells exposed to allogeneic TLR9activated plasmacytoid dendritic cells (pDCs), but not B-cells, leads tothe generation of iTregs (Moseman et al., 2004, J Immunol 173:4433-42),CD4⁺25⁺FoxP3⁺ suppressor cells, a process dependent upon the enzymeindoleamine 2,3-dioxygenase (IDO) that catabolizes tryptophan. Adding 50μM trypt catabolites (kynurenines: KYN) to allogeneic B-cell culturesgenerated potently suppressive iTregs. Because obtaining high numbers ofnon-malignant B-cells or pDCs from leukemia/lymphoma patients would bedifficult, low trypt/KYN conditions in an artificial antigen-presentingcell expansion system was examined. In considering strategies for iTreggeneration, experiments were designed to focus on molecular featurespreferentially associated with nTregs vs naïve or T effector cells.Compared to non-Tregs, nTregs have hypomethylated DNA and upon T cellreceptor signaling, FoxP3 binding to CD25, GITR, CTLA4 increases histoneacetylation (Chen, et al., 2006, J Biol Chem 281:36828-34). nTregs arein a state of hypomethylation and histone acetylation. In leukemias,tumor suppressor genes are inactivated by oncogenic transformation anddemethylating agents (decitibine) and HDACi (SAHA, vorinostat) are usedfor treatment (O'Connor et al., 2006, J Clin Oncol 24:166-73;Garcia-Manero et al., 2008, Blood 111:1060-6; Issa et al., 2005, J ClinOncol 23:3948-56; Issa et al., 2004, Blood 103:1635-40).

Without wishing to be bound by any particular theory, the aforementionedagents are useful for large-scale iTreg generation. mTOR/Akt signalingis upregulated in myeloid leukemias and rapa is used for AML/MDS therapy(Yee et al., 2006, Clin Cancer Res 12:5165-73). Adding rapa toperipheral blood Treg cultures favors iTregs and nTregs due to higherdependency of non-Tregs on mTOR/p-Akt for proliferation and survival(Zeiser et al., 2008, Blood 111:453-62; Battaglia et al., 2005, Blood105:4743-8; Coenen et al., 2006, Blood 107:1018-23; Haxhinasto et al.,2008, J Exp Med 205(3):565-74). The results presented herein demonstratethat combinatorial approaches of low trypt/KYN, decitbine, SAHA and rapamay be used for converting non-Tregs into Tregs.

Studies are performed to determine whether CD25+ T cells should bedepleted (w/ CD25 beads) and whether CD45RA positive selection is neededby comparing results of CD4⁺, CD4⁺25⁻, CD4⁺45RA⁺ and CD4⁺25⁻CD45RA⁺input T cells using the preferred iTreg protocol. CD45Ra beads oranti-CD45Ra mAb may be used in conjunction with Miltenyi.

Expansion procedures include using a bead- or cell-based artificialantigen-presenting cell system (CD3/28 beads vs KT64/86 cells). T cellsare cultured at 1×106/ml in the appropriate culture vessel andstimulated with IL-2 (300 U/ml) and anti-CD3/28 beads or OKT3-loaded,irradiated KT64/86 cells. Cultures are diluted 1:1 on day 3 withmedia/IL2 to maintain T cell:APC clusters. On day 7, cultures are splitinto larger vessels and re-plated to 0.5×106/ml thrice weekly.

The next set of experiments is a phase I dose escalation trial ofperipherial blood nTregs. Patients with malignancy and HLA genotypicidentical donors are subjects for the phase I trial. The trial isdesigned to include standard GVHD prophylaxis agents previously shownnot to have a deleterious effect on Treg function in vitro. With thehigh numbers of iTregs generated from the combinational approachdiscussed herein, a 3rd infusion on day 28 can be readily incorporatedinto this platform.

Ultimately, the methods discussed herein are useful for manufacturingsufficient iTregs for about two to three infusions aftercryopreservation after the completion of in vitro and in vivo potencyand stability assays. Once the maximal tolerable dose is determined andin vivo activity is verified, it is anticipated that the next generationof clinical trials will eliminate calcineurin-inhibitors, steroids andother agents, as post transplant immunosuppression (eg. rapa, MMF).

Example 7 Restimulation of iTreg

The next set of experiments was designed to determine whetherre-stimulation of iTreg increases the percentage of Foxp3++ cells. nTreg(CD4⁺25⁺⁺) and CD4⁺25⁻45RA⁺ cells were purified from peripheral blood(PB) using magnetic beads and the cells were stimulated with anti-CD3loaded KT64/86 cells (K562 cells modified to express an Fc receptor CD64and CD86). nTreg were cultured in the presence of IL-2 (300 U/ml) andRapamycin (109 nM) while CD4⁺25⁻ cells were cultured with IL-2 alone, orwere induced to become Treg by the addition of Decitibine (5 μM at day7) and Raparnycin (109 nM at day 7). Portions of the CD4⁺25⁻45RA⁺ andDecitibine iTreg cultures were also re-stimulated on day 7 with anti-CD3loaded KT64/86 cells. After a total of 14 days, cells were harvested andthe expression of CD4, CD25, and Foxp3 was measured using flowcytometry. FIG. 6 summarizes the results in the context of percentage ofCD4+ gated cells expressing low (Foxp3+) or high (Foxp3++) levels ofFoxp3. It was observed that re-stimulation of CD4+25−CD45RA+ cells doesnot significantly effect Foxp3 levels. However, the percentage of cellsthat were Foxp3++ was significantly increased in the Decitibine iTreggroup following re-stimulation.

The next set of experiments was designed to determine whether TGFβ couldsynergize the effects of Decitibine to induce Foxp3 expression in thecells. Briefly, nTreg (CD4⁺25⁺⁺) and CD4⁺25⁻45RA⁺ cells were purifiedfrom peripheral blood using magnetic beads and stimulated with anti-CD3loaded KT64/86 cells. nTreg were cultured in the presence of IL-2 (300U/ml) and Rapamycin (109 nM) while CD4⁺25⁻ cells were cultured with IL-2alone, or were induced to become Treg by the addition of Decitibine (5μM at day 7) in the presence or absence of TGFβ (10 ng/ml, day 0). Allsamples were re-stimulated on day 7 with anti-CD3 loaded KT64/86 cells.After a total of 14 days, cells were harvested and expression of CD4,CD25, and Foxp3 was measured using flow cytometry. It was observed thatwhile stimulation of CD4⁺25⁻45RA⁺ cells and Decitibine treatedCD4⁺25⁻45RA⁺ cells with anti-CD3 loaded KT64/86 cells resulted in someFoxp3 expression, Foxp3 expression was significantly increased whenCD4⁺25⁻45RA⁺ cells were treatment with both Decitibine and TGFβ,especially the Foxp3++ cells (P≦0.01 and ≦0.002 for unmanipulated vs.cells treated with Decitibine or Decitibine/TGFβ, respectively). SeeFIG. 7.

The next set of experiments was designed to determine whether Tregsinduced with Decitibine and TGFβ secrete IFNγ. nTreg (CD4+25++) andCD4+25−45RA+ cells were purified from PB using magnetic beads and werestimulated as discussed elsewhere herein. After 12 days, cells werewashed, resuspended in fresh media and incubated for an additional 2days, after which the cells were removed by centrifugation and thesupernatants assayed for IFNγ, FIG. 8 indicates that Tregs induced withDecitibine and TGFβ do not secrete IFNγ. The line at 15 pg/ml in FIG. 8represents the sensitivity of the assay. Supernatants were also assayedfor IL-17, but significant amounts of this cytokine were not detected(not shown).

The next set of experiments were designed to evaluate the effects ofDecitibine and Decitibine/TGFβ induced Tregs in a xenogeneic model ofGVHD. Briefly, nTreg (CD4+25++) and CD4+25−45RA+ T cells were purifiedand expanded with cell based aAPC (KT64/86 at day 0 and 7) and culturedin the presence of Decitibine or Decitibine/TGFβ as discussed elsewhereherein. On day 14, nTreg, Decitibine iTreg, or Decitibine/TGFβ iTregwere co-transferred with allogeneic PBMC into clodronate-treated,irradiated Rag2^(−/−), γ_(c) ^(−/−) mice. FIG. 9A depicts flow phenotypeat Day 4 of the number of CD4+Foxp3+ per μl blood for each of the celllines used. FIG. 9B represents a Kaplan-Meyer survival curve showingincreased survival of clodronate-treated, irradiated Rag2^(−/−), γ_(c)^(−/−) mice receiving human PBMC±groups of Treg in a 3:1 ratio (i.e. 30-and 10×106 cells, respectively). n=10, 8, 7 and 6 for groups PBMC,nTreg, Decitibine iTreg and Decitibine/TGFβ iTreg respectively. P≦0.05for each Treg treated group compared to PBMC.

The next set of experiments were designed to determine the feasibilityof large-scale production of functionally suppressive, Foxp3+ inducedTreg using Decitibine, or Decitibine plus TGFβ. Table 1 is a summary ofthe results from a representative large-scale purification and in vitroexpansion experiment of Treg induced with Decitibine (n=3) or Decitibineplus TGFβ (n=4) in comparison to nTreg purified from umbilical cord(n=15) or peripheral blood (n=5). iTreg expansion was performed using aGMP-compliant cell line (KT64-86), and generated increased numbers ofFoxp3+ cells (Total cell number * % CD4+25+Foxp3+) as compared to theclinical scale productions of umbilical cord or peripheral blood nTregexpanded with anti-CD3/28 beads (5- and 2-fold, respectively).

Purification Expansion Initial Purified Initial % Purified Final % cellcell CD4+, % CD4+, cell CD4, number number 25++, 25++, Expansion number*CD25+, Suppression Source Cell type (×106) (×106) FoxP3+ FoxP3+ (Fold)(×106) Foxp3+ index UCB nTreg 2171 ± 258 7.5 ± 2.3 3.6 ± 1.4 45 ± 5 583± 127 6960 ± 2571 52 ± 8 1:2-1:64 (73% @ 1:4) PB nTreg 12,800 196 2.9 4169   14,200  62 1:4-1:64 (±1350) (±20) (±0.6) (±6) (±22)   (±6160) (±7)(70% @ 1:4) Decitibine 338 N/A N/A 64.5 21801   36.6 1:2-1:8 iTreg*(±103)  (±3.1) (±1048) (±4) (48% @ 1:4) Decitibine/TGFβ 82.2 27784 711:2-1:8 iTreg* (±25.2)  (±12753)  (±17)  (62% @ 1:4) *Calculated totalcell number from experiments initiated with 2 × 109 PBMC.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety.

While this invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims are intended to be construed to include all such embodiments andequivalent variations.

1. A method of generating an inducible T regulatory cell (iTreg), saidmethod comprising contacting a non-Treg with an agent capable ofconverting said non-Treg into an iTreg, wherein said iTreg isimmunosuppressive.
 2. The method of claim 1, wherein said agent isselected from the group consisting of a breakdown product of tryptophan,an analog of a metabolic breakdown product of tryptophan, a tryptophancatabolite, a demethylating agent, a histone deacetylase inhibitor(HDACi), an mTOR inhibitor, and any combination thereof.
 3. The methodof claim 1, wherein said non-Treg is selected from the group consistingof CD4⁺, CD4⁺CD25⁻, CD4⁺CD25⁻45RA⁺ cell, and any combination thereof. 4.The method of claim 1, wherein said iTreg is CD4⁺CD25⁺.
 5. The method ofclaim 1, wherein said non-Treg is isolated from a sample obtained fromleukopheresis products, bone marrow, lymph tissue, thymus tissue, spleentissue, or umbilical cord tissue.
 6. The method of claim 2, wherein saidtryptophan catabolite is kynurenines.
 7. The method of claim 2, whereinsaid mTOR inhibitor is rapamycin.
 8. The method of claim 2, wherein saiddemethylating agent is selected from the group consisting of5-aza-2′-deoxycitidine (decitibine), 5-Azacytidine, and any combinationthereof.
 9. The method of claim 2, wherein said HDACi is selected fromthe group consisting of trichostatin A (TSA), suberoylanilide hydroxamicacid (SAHA), and any combination thereof.
 10. The method of claim 1,further comprising contacting said non-Treg with a bead or artificialantigen-presenting cell (aAPC) expansion system prior to orsimultaneously with said agent.
 11. The method of claim 10, wherein thebead comprises anti-CD3 antibody and anti-CD28 antibody.
 12. The methodof claim 1, wherein said non-Treg is further contacted with TGFβ. 13.The method of claim 1, wherein said iTreg is positive for Foxp3.
 14. Themethod of claim 1, further comprising contacting said iTreg with a beador artificial antigen-presenting cell (aAPC) expansion system subsequentto contacting said non-Treg with said agent.
 15. The method of claim 14,wherein said aAPC comprises an immune stimulatory ligand and at leastone co-stimulatory ligand on its surface.
 16. The method claim 15,wherein said stimulatory ligand is a polypeptide selected from the groupconsisting of a major histocompatibility complex Class I (MHC class I)molecule loaded with an antigen, an anti-CD3 antibody, an anti-CD28antibody, an anti-CD2 antibody, and any combination thereof.
 17. Themethod of claim 15, wherein said co-stimulatory ligand is selected fromthe group consisting of CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2,4-1BBL, OX40L, ICOS-L, ICAM, CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB,HVEM, lymphotoxin beta receptor, ILT3, ILT4, 3/TR6, a ligand thatspecifically binds with B7-H3, and any combination thereof.
 18. A methodfor inhibiting alloreactive T cells, the method comprising contactingsaid alloreactive T cells with an effective amount of iTregs.
 19. Amethod for inhibiting cytotoxic T-lymphocyte (CTL) activity, the methodcomprising contacting a cytotoxic T-lymphocyte with an effective amountof iTregs.
 20. A method for generating an immunosuppressive effect in amammal having an alloresponse or autoimmune response, the methodcomprising administering to said mammal an effective amount of iTregs.21. The method of claim 20, wherein said mammal having an alloresponseor autoimmune response follows tissue transplantation, and wherein themethod further comprises suppressing, blocking or inhibitinggraft-vs-host disease in the mammal.
 22. The method of claim 20, whereinsaid mammal is a human.
 23. A method for preventing an alloresponse oran autoimmune response in a mammal, said method comprising administeringto said mammal, prior to onset of an alloresponse or autoimmuneresponse, an effective amount of iTreg to prevent said response.
 24. Themethod of claim 23, wherein said mammal is treated prior to, at the timeof, or immediately after tissue transplantation, and wherein the methodfurther comprises preventing onset of graft-vs-host disease in saidmammal.
 25. The method of claim 24, wherein said mammal is treated priorto, at the time of, or immediately after tissue transplantation, andwherein the method further comprises blocking rejection of thetransplanted tissue in the mammal.
 26. The method of claim 23, whereinsaid mammal is a human.
 27. A method of treating a transplant recipientto reduce in said recipient an immune response against the transplant,the method comprising administering to a transplant recipient, aneffective amount of iTregs to reduce an immune response against theantigen.
 28. The method of claim 27, further comprising administering tosaid recipient an immunosuppressive agent.
 29. The method of claim 27,wherein said iTregs are administered to the recipient prior to saidtransplant, concurrently with said transplant, or subsequent to thetransplantation of the transplant.